BIOLOGY 204/205 Advanced Genetics Laboratory
TABLE OF CONTENTS
Introduction………………………………………………………………..…..………….…p.2
MODULE 1: Recombinant DNA…………………………………………...…….…..p.11
MODULE 2: RNA Interference………………………………………………………..p.25
MODULE 3: Proteomics………………………………………………………………….p.38
Appendix A: Solutions Guide……………..………………………………....……...p.47
General guide…………………….……………………………p.47
MODULE 1………………………….…………………………..p.49
MODULE 2…………………………………….………………..p.53
MODULE 3…………………………………….………………..p.54
Appendix B: Sterile Techniques.…………………………………………..….......p.58
Appendix C: Spread Plate Technique………………………………………….....p.58
Appendix D: Pipette Use…………………………………………………………….....p.59
Appendix E: Pipetting Exercise Pierce BCA Assay………………..…………p.61
Appendix F: GST Plasmid Map………………………………………………………..p.62
Appendix G: L4440 RNAi Plasmid Map …………...……………….…….....…p.63
Appendix H: Streak Plate Method………………………..………….……......…p.64
Appendix I: Frequently Used Ladders.…………………………….……………..p.65
Appendix J: Southern Blot setup………………………….………..…..………....p.66
Appendix K: PCR Chart for 1.17 ………………..…………………..……….….....p.67
Appendix L: Chart for Making Sucrose …………………………..………………p.68
Appendix M: Sucrose Gradient Tubes after Ultracentrifugation …...p.69
Appendix N: Protein Gel Plate Setup ….……………………………………...…p.70
Appendix O: Pierce BCA Protein Assay for Module 3.………………..…..p.71
Appendix P: Assembling Protein Gel for Running Gels…..………........p.72
Appendix Q: Graphing Protein Data in Excel…………………………..……..p.73
Appendix R: RPM to G-force Conversions……………………..………..……..p.74
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BIOLOGY 205
Advanced Genetics Laboratory Spring Semester
--- Introduction --Course Goals:
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Master techniques of molecular biology
Trouble shoot based on the student's knowledge of organic chemistry and biology
Practice hypothesis development
Improve written expression
Learn collaboration with lab members
Keep a good laboratory notebook
Cultivate an ability to develop protocols and experiments to test a hypothesis
Become facile in graphing and spreadsheets in the analysis of data
Become facile in statistical analysis of spreadsheet data
Gain practical knowledge that will help with careers and jobs in research
Put into practice the theoretical information that the student has learned through the
100 levels
Become inspired to go on in biological science as a career
Biology 204/205 Advanced Genetics Laboratory
--- Introduction --Biology 205 is a four credit course. Those of you whom are new will complete the experiments
outlined in Module 1 and 3. Those of you whom have taken Biology 204 will complete the
experiments outlined in Module 2 and 3. In both Modules 1 and 2, you will write a laboratory
report. For Module 3, you will be asked to develop a hypothesis and experiments you would
use to test your hypothesis based on identified proteins. The final exam will be a class
presentation that you will present during finals with your lab partner.
The emphasis of Biol 205 is on experimental design, techniques, data gathering and analysis.
Work at the bench is given priority over work in a lecture setting. The modules are designed to
approach real situations in ongoing research projects. Therefore, the modules are not
necessarily designed to be finished in three hours. A few labs will run long, taking 5-6 hours to
finish. A few labs will be relatively short. Students are expected to come into lab outside of the
scheduled class time, usually at their own convenience, to perform short manipulations.
Sometimes an experiment does not work and it has to be repeated. Coming to class well
prepared and following directions carefully will cut down on potential mistakes!
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Module 1 Recombinant DNA/Bacterial Transformation
This module gives you some of the experience you would receive if you were to sub-clone a
gene as a part of your research. That is, once you transform a bacterial line with the plasmid
that you isolate, you will need to demonstrate that you have made the transfer of the correct
gene.
Goals:
1. Purify a plasmid and transform E. coli with the plasmid.
2. Demonstrate that the transformants carry the plasmid by characterizing the
transformants’ phenotypes.
3. Analyze the size of the DNA plasmid in a cracking gel.
4. Hybridize with the original plasmid in a Southern blot.
5. Amplify the gene inserted into the plasmid by PCR.
6. Sequence part of the plasmid.
Module 2 RNA Interference (RNAi)
In this module, a specific target gene product of Paramecium tetraurelia is depleted using an
RNA Interference (RNAi) feeding method. You will isolate total RNA from test and control
paramecia then determine if there has been down regulation of the gene product in the RNAi
treated population compared to the control treated population.
Goals:
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Design RNAi plasmid and primers.
Test primers for amplification of endogenous mRNA (cDNA) by PCR.
Isolate RNA from harvested paramecia cells.
Synthesize cDNA from the mRNA collected (Reverse transcriptase PCR).
Determine the level of target endogenous mRNA by semi-quantitative Reverse
Transcriptase- Polymerase Chain Reaction (RT-PCR)
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Module 3 Proteomics
In this module you will compare the proteins found in the pure ciliary membrane of wild type
and mutant paramecia. You will run a one dimensional polyacrylamide gel, and cut the lanes of
the gel from both the wild type and mutant samples. The class will then compare the proteins
identified in the two samples as well as compare the abundance of proteins between the
samples.
Goals:
1. Use ultracentrifugation to isolate pure ciliary membranes from wild type and mutant
cells.
2. Conduct a BCA Protein Assay to determine the concentration of proteins in samples
provided to you.
3. Using SDS-PAGE, run a one dimensional gel and stain with Coomassie blue to visualize
differences in protein content of cilia types.
4. Cut out bands of interest from gel and prepare samples for mass spectrometry by
trypsinizing proteins.
5. Analyze results from mass spec.
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Biology 205 Advanced Genetics Laboratory
Grading Policy
Your grade will be based on the following components, each with approximately equal weight
(20%):
1. Performance in laboratory (see more detailed list below).
2. Discussion of experiments and participation in student-led review sessions.
3. Notebooks (these will be checked approximately every other week).
4. Laboratory report (50% of this portion for the rough draft; 50% for the final report).
5. Final presentations.
*NOTE*: 10% will be deducted from the grade per day the assignment is late –
for each 24 hours the assignment is late, another 10% is deducted.
Plagiarism
Plagiarism is taken very seriously and can be an expellable offense. The University of
Vermont has defined plagiarism as the following:
“All ideas, arguments, and phrases, submitted without attribution to other sources must
be the creative product of the student. Thus, all text passages taken from the works of
other authors (published or unpublished) must be properly cited. The same applies to
paraphrased text, opinions, data, examples, illustrations, and all other creative work.
Violations of this standard constitute plagiarism.” (University of Vermont, Vice Provost
for Student Affairs, Policy V. 2.7.7)
If you are unsure of what constitutes plagiarism or if something is considered
“paraphrased”, please come and see Dr. Valentine for further explanation. Simply placing a
citation and still copying the text or changing a few words within a paragraph that has been
copied still constitutes plagiarism.
For more resources and information on plagiarism, please see the following:
University of Vermont policy:
http://www.uvm.edu/policies/student/acadintegrity.pdf
Harvard explanation with specific examples:
http://isites.harvard.edu/icb/icb.do?keyword=k70847&pageid=icb.page342054
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Performance in Laboratory Guidelines
1. Accuracy and quality of results.
2. Lab citizenship includes:
a. Arriving on time and staying until work is complete.
b. Coming in willingly outside of class when necessary.
c. Supporting your lab partner(s); being present/engaged during experiments.
d. Following the safety rules, cleaning up, properly labeling your samples, putting
materials away, etc.
3. Working well and cooperatively with lab partner(s).
a. Simulates collaborative behavior.
b. Behavior is professional.
4. Arriving prepared for the day’s methods, reading the manual ahead of time (*see
laboratory notebook below).
a. Working efficiently.
b. Following through.
5. Attitude and willingness to participate in class/discussions/experiments.
Laboratory Notebook Guidelines
We expect you will come prepared for class by writing the procedure for the day ahead of time
in your laboratory notebook. When changes occur, as in any research lab, they can be marked
and initialed in your laboratory notebook. You are expected to have your lab notebook on your
work bench, not your lab manual (the manuals can be referred when necessary).
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Hard cover, bound notebook; no loose-leaf notebooks.
Legibly record in blue or black ink – do not photocopy/print and paste the lab manual
into your notebook.
Number all pages.
Date all entries.
Name, course number and email address should be on front cover.
Reserve 3 pages at the beginning for the table of contents; keep up to date.
Mistakes should be crossed out with a single line through the entry then initialed.
Do not skip pages, do not rip pages out.
Unused portions of a page should have a diagonal line drawn through the blank portion.
Each experiment should begin on a new page.
All data, calculations and graphs should be entered directly into the notebook.
Your notebook should provide enough detail so that another Advanced Genetics student could
pick it up and repeat your procedure by following your entries. You should include all of the
following information for each section:
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Objective
Who did the experiments (some procedures are done for you)
Hazardous for that module
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All recipes for solutions used (Appendix A)
Why were the procedures done
Results
Conclusions/discussion
A sample lab notebook is available in the lab for you to look at.
Laboratory Report Guidelines
You will be asked to write a formal report on Module 1 (newcomers) or Module 2 (returning
students). Included should be an abstract, introduction, methods, results, and discussion
sections. To aid your preparation of this report, you should go to the library early in the
semester and find a short article from Genetics from the last 5 years (download a pdf version or
photocopy from a paper journal, the library has both formats). The format in Genetics is
appropriate for your report. Below is a description of the content and length of each section.
The report should not exceed 12 pages in length (exclusive of the references). The font size and
spacing is outlined in each section below. Each section should be started with its section name,
in bold type. At the head of the report, you should provide a title that indicates which exercise
you are writing about and your name. Whenever possible, you should strive to write succinctly
and in the active voice. The methods section should be in the past tense. Please include page
numbers.
Abstract: (12 point font, single spaced). The abstract must not exceed 250 words and should
summarize the experiment and outcomes.
Introduction: (12 point font, double spaced). The introduction provides an overview of what
the report is about, including why the exercise was done (the goal of the exercise) and an
explicit statement of the hypothesis or hypotheses being tested. Background information about
the biology underlying the techniques should be included in the introduction. Please be sure to
look at the seminal work introducing the techniques you have used; be sure to cite them (see
References). Recommended length: 1.5 -2 pages.
Methods: (10 point font, single spaced). The methods section must be detailed enough to
allow the reader to repeat the experiments. It should be written in the past tense. You do not
need to repeat the detailed description of the protocols in the laboratory manual. Look at
published articles to see how the Materials and Methods sections are written. Some charts and
tables can be included in an Appendix of your report (i.e., PCR set up for Module 1, or the
making of cDNA for Module 2). Recommended length: 3 pages.
Results: (12 point font, double-spaced; Figure legends: 10 point font, single spaced). The
results section reports upon what happened during the exercise. You must include images of
the final gels (gels or unsuccessful results can be included in the Appendix when necessary).
Figures with figure legends should be embedded in the text. Provide, in tabular form, other
measurements and data you collected. Each figure should have a brief descriptive caption
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(Figure legend), and each table should have a title. However, it is not sufficient to simply insert
these figures and tables. You must interpret your results in the text of the section, with
references to the appropriate figure or table (Format: Fig. 1, Table 2A). Recommended length:
2-3 pages.
Discussion: (12 point font, double spaced). In the discussion, you should briefly re-introduce
the main goal or hypothesis presented in the introduction, and then describe how your results
are related to the goal or hypothesis. In subsequent paragraphs, you should discuss any failures
to obtain results, and describe what you believe happened and what you would do differently
to correct each problem. This is your opportunity to show how well you understand the
molecular processes underlying the protocols! Recommended length: 2 pages.
References: There should be at least 5 references cited in the lab report. You are expected to
look for some of the original experiments that explored the techniques you are using. Typically,
the references are cited in the Introduction, but can be used elsewhere. References should be
cited in the text using APA format (Author, date) with a proper reference list at the end.
General Lab Safety Rules:
1. Disinfect your bench top with a 10% bleach solution when you arrive and when you
finish lab each day.
2. Wear gloves and avoid touching face and/or hair during an experiment.
3. Wash your hands before you leave lab.
4. Do not eat, drink, smoke, chew gum or apply cosmetics while in lab.
5. Dispose of all used materials as directed.
6. Keep aisles clear – push in your chairs.
7. Wipe all spills immediately. Inform lab tech and/or TA if you spill ANYTHING.
8. Dispose broken glass in the appropriate receptacle. Inform lab tech and/or TA that you
have broken glass.
9. Wear closed-toed shoes.
10. Tie back long hair.
11. Wear pants.
12. Avoid wearing baggy, loose clothing or accessories that can interfere with your
experiment and/or may catch on fire.
13. Extinguish burners as soon as you finish using them.
14. All Chemical Safety and MSDS information is located in the binder on the back of the
door.
15. If you are unsure about a procedure, Please ask.
16. Cell phone, tablets, iPads, etc. are to be used only in the hallway. Please leave cell
phones outside the lab room. Points will be deducted for using cell phones and devices
in the lab.
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Laboratory Exercise for First Day of Lab: Pipetting
Background:
1. Acquaint yourself with the various denominations of pipettors in an attempt to avoid
mistakes, particularly when working under time pressure. (See Appendix D.)
2. The height of the fluid in the glass pipettes is measured at the bottom of the meniscus
while the pipette is being held vertically.
3. Never put a pipette back into a sterile container.
4. Do not handle the lower part of the pipette.
Serial Dilutions: Serial dilutions allow you to dilute a sample many fold by making a series of
small dilutions.
Standard Dilution Steps: Unless special circumstances demand it, the following are the only
dilution steps that are used (For convenience and error avoidance in performing the
accompanying arithmetic): 10, 20, 50 and 100.
10X
1:10
0.1 mL/0.9 mL
100 µL/900 µL
20X
1:20
0.1 mL/1.9 mL
50 µL/950 µL
50X
1:50
0.1 mL/4.9 mL
20 µL/980 µL
100X
1:100
0.1 mL/9.9 mL
10 µL/990µL
Pipette Exercises
Pipette Exercise #1
Determine and record the pipettor(s) best suited for each of the measurements listed below.
Volume
10 µL
2 µL
598.6 µL
Pipettor(s)
Volume
25µL
0.015 mL
0.200 mL
Pipettor(s)
Volume
0.0963 mL
183 µL
201.4 µL
Pipettor(s)
Pipette Exercise #2 : Pierce Protein Assay
Use the chart provided (See Appendix E) to develop a standard curve using BSA standards and
to determine your unknown protein concentration. You will be setting up duplicates of
Appendix E, running two sets of protein assays and averaging the OD values to calculate the
unknown protein concentrations.
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1. Obtain unknown protein samples from your TA, place 100 µL of the unknowns into the
appropriate tubes.
2. Add BSA and dH2O according to the directions in Appendix E to the remaining tubes.
3. Only after all known and unknown tubes are prepared should you make the dye
solution: Use Solutions A and B from the Pierce BCA Protein Assay Kit. They should be
mixed 50:1 (A:B). Show the calculations of the amount of dye you plan to make to the
TA. You should plan on having a few extra tubes. Mix the dye in the container provided.
4. Add 2 mL of the dye to each one of your unknown samples and standards.
5. Incubate tubes at 37C for 30 minutes.
6. Get OD values for standards and samples. The TA will assist in the operation of the
spec.
7. Place 1 mL your first standard (blank) into a clean cuvette and use to blank the spec.
 Rinse the cuvette between each sample using dH2O.
 You may use more than one cuvette.
 Make sure to dry the outside of the cuvette with a Kimwipe™.
 Handle the cuvette only on the top, smudges can interfere with the absorbance.
8. Read OD at 562 nm.
9. Repeat to obtain an absorbance for each standard and unknown sample. Do the
readings for one complete set of standards and unknowns, then the second set of
standards and unknowns.
Do not blank the spec between each standard and unknown. Once the spec is blanked,
do not blank again.
10. Find the average OD’s of each standard and sample duplicates.
11. Establish a standard curve using the OD values obtained with your BSA standards (See
Appendix Q): graph Concentration (x-axis) vs. OD (y-axis) on the computers. Do not
force the line through 0. Be sure to properly label your X- and Y- axis. Using the
equation from the slope, calculate the unknown protein concentrations of your
unknowns.
Pipette Exercise #3
Using the p1000 or the p200 pipettors, perform the following:
1. Set the pipettor to its maximum volume.
2. Using water at room temperature, carefully pipette the water onto a weigh boat that
you have tared (re-zeroed).
Room temperature water has a density of approximately 1 g/mL or 1 g/L. Therefore, you can
determine the accuracy of your pipetting, e.g., 1000 L of water will weigh 1 g. Repeat the
pipetting until you feel that you are reasonably accurate. Then record the weights of five
successive pipettings. Determine the mean and standard deviation associated with your
measurements.
Complete the following conversions:
1 L
=_______mL
0.001 L
10 L =_______mL
0.11 L
100 L =_______mL
0.01 L
1000 L =_______mL
1L
=________mL
=________mL
=________mL
=________mL
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Module 1
Recombinant DNA
Please refer to page 3 for introduction
**Note: The E.coli cell line used in this module is K12. The K12 bacteria cells are to be
transformed with the GST plasmid (see Appendix F).
** Note: For conversions of all centrifuge speeds from RPM to RCF or × g, See Appendix R.
1.0 Overnight (ON) Bacterial Culture (Done for you)
1. The lab tech will add 2.5 mL of cells previously grown ON to 100 mL LB-AMP medium
(per group).
2. The cells will grow while shaking at 37°C ON.
1.1 Isolation of Plasmid DNA
HAZARDOUS CHEMICAL INFO:
-Salt-Saturated Phenol (SS Phenol) is to be used only while wearing gloves under the hood.
Dispose of all pipets and liquid waste containing SS Phenol in appropriate disposals.
-Chloroform: Isoamyl Alcohol (24:1) (C:IA) is to be used only while wearing gloves under the
hood. Dispose of all pipets and liquid waste containing C:IA in appropriate disposals.
1. Transfer 100 mL of the overnight bacterial culture (Transformed E. coli) to a large,
sterile, screw top centrifuge bottle and harvest the bacteria by centrifuging at 5000 rpm,
4°C for 10 minutes in the Beckman J2-21.
2. Decant the supernatant broth into the waste jar.
3. Resuspend the bacterial pellet in 5 mL of Solution I containing 5 mg/mL lysozyme.
4. Transfer to a 30 mL polycarbonate screw top Oakridge centrifuge tube. Let stand at
room temperature for 5 minutes.
5. Add 10 mL of freshly made Solution II. Place the cap on the tube and mix the contents
by inverting the tube several times, mixing gently. Let stand on ice for 10 minutes.
6. Add 8 mL of ice-cold 3M potassium acetate (pH 4.8). Fill tubes only ¾ full. Screw on the
cap and mix by inverting. Let stand on ice for 30 minutes. (Total of 22 – 23 mL.)
7. Balance the tubes before centrifugation.
8. Centrifuge in the Beckman J2-21; 15,000 rpm, 4°C for 20 minutes. The genomic DNA
and bacterial debris should form a tight pellet at the bottom of the tube.
9. Being very careful not to disturb the pellet. Use a 10 mL pipet and transfer all to a 30 mL
glass screw top tube. Only take clear supernatant.
10. Add 1 mL of heat treated RNase A to the tube. Please use all of the RNase A provided.
11. Incubate at 37°C for 10 minutes in Innova 4000 then invert 2 to 3 times. Incubate and
additional 10 minutes in the Innova 4000.
12. Using glass pipettes, divide your original solution as necessary into 3 or 4 conical tubes.
In the fume hood, add one volume of SS phenol using glass pipettes. (Note the yellow
color which helps you identify the phenol phase in the next step.) Your tubes can be no
more than 2/3 full (12 mL) including the addition of the phenol.
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SAFETY NOTE: Phenol can cause severe burns to skin and damage clothing. Gloves and
safety glasses should be worn when working with phenol. All manipulations should
be carried out in the fume hood. A glass receptacle is available exclusively for
disposing of used phenol and chloroform.
13. Vortex the conical tube and contents with lids on for 1 minute; be sure the contents are
thoroughly mixed. Make sure the tops of the conical tubes are screwed on tightly to
ensure that no leaking will occur. Centrifuge for 1 minute at 2800 rpm using the
Eppendorf Centrifuge 5702.
14. Transfer the non-colored upper aqueous phase to a fresh conical tube. Do not take the
interface, which contains denatured protein. In the hood, add 1 volume of chloroform:
isoamyl alcohol (24:1) – the same amount as the phenol you added. Vortex 1 minute
and centrifuge 1 minute at 2800 rpm.
15. Transfer the upper, aqueous layer to a fresh 30 mL glass tube and add 2.5 volume of
cold 95% ethanol, using glass pipettes. You need to calculate how much total liquid will
be in each 30 mL glass tube. The tube cannot be more than 2/3 full, (20 mL) so you may
have to use more than one 30 mL glass tube. Make your calculations before adding the
ethanol!
16. Mix and allow it to precipitate in the -80°C freezer overnight.
Next Day (This will require 1 to 1.5 hours, please plan accordingly)
17. Balance your tubes along with their rubber sleeves.
18. Recover the DNA by centrifuging the tube at 4°C in the Beckman J2-21 at 9500 rpm for
30 minutes.
19. Discard the supernatant into a waste container. The pellet will look like a whitish residue
on the side of the tube.
20. Resuspend the pellet with 1 mL 70% ethanol by pipetting up and down onto the sides of
the tube. Try to resuspend the entire pellet to increase your plasmid yield. Transfer the
resuspention from the first tube to the next tube until all pellets are resuspended and
pooled together. Transfer the solution into one sterile 1.5 mL microfuge tube.
21. Microcentrifuge for 5 minutes at 14,000 rpm. Pipet out the ethanol; add 1 mL more of
70% ethanol to wash the pellet and vortex for 30 seconds. Spin at 14,000 rpm for 5
minutes.
22. Discard the ethanol; dry the pellet using the SpeedVac in the basement. Give your
sample to the TA/Lab tech to be properly dried for 15 – 20 minutes.
23. Dissolve the pellet in 0.3 mL TE. Aliquot 100 L to each of 3 microcentrifuge tubes
(properly labeled!).
24. Store at -20°C.
1.2 Agarose gel to confirm isolation of the plasmid
HAZARDOUS CHEMICAL INFO:
-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and
dispose of everything that has contacted EtBr in the appropriate solid waste container.
-UV light is very harmful if looked at directly. When viewing your gels on the UV light box be
sure to wear a protective face mask, or place the shield on top of the box before turning on
light.
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1. Dissolve 0.35 g agarose in 50 mL 1X TAE buffer to make a 0.7% gel.
2. Microwave on high for 1 minute.
3. Swirl the flask and make sure all of the agarose is dissolved. If not, microwave until it is.
Remove flask with a hot mitt.
4. Place the running tray into the gel-casting tray. Add comb.
5. Cool agarose slightly; approximately 5 – 10 minutes, swirling occasionally. Slowly pour
agarose into the farthest corner from the comb in the gel casting set up. Try to avoid
bubbles! If bubbles appear remove them with a pipette tip. Let cool until opaque
(approximately 20 minutes).
6. While your gel is setting, thaw out one tube of your plasmid DNA on ice.
7. Your TA will have determined your DNA concentration. Based on your concentrations,
dilute your plasmid DNA sample as necessary in TE buffer.
8. Just before you are ready to load the gel, heat the λ Hind III marker (See Appendix I)
for 7 minutes in the 65°C hot block – Place on ice immediately.
9. Once your gel is set, remove it from the casting tray. Place it in the running tray, with
the comb still set. Cover the gel with 1X TAE. Gently remove the comb. Removing the
comb last will ensure that your wells do not collapse.
10. Mix 4 µL of 6X DNA loading buffer with 20 µL plasmid or diluted plasmid DNA on a piece
of Parafilm or in a microfuge tube.
11. You must also prepare 1X DNA loading buffer for all empty lanes. Each empty lane
should get 20 µL 1X DNA loading buffer.
12. Be prepared to load the gel quickly—you do not want your DNA to diffuse into the
running buffer.
13. Load 24 µL of plasmid DNA sample and 20 µL of λ Hind III marker in the wells; put the lid
on the box so that the DNA will run toward the red electrode.
14. Run the gel at 100V for ~1 hour.
15. Stain the gel for approximately 15 minutes in ethidium bromide, followed by a brief
rinse in water.
16. Examine the gel on the UV light box. If the ladder is not visible or is faint, place the gel
back into the stain. When you feel that your gel is properly stained, take a picture to
document your results.
17. Leave the gel in destain to be discarded later.
18. Rinse electrophoresis unit with RO water after use so the buffer does not dry on the
electrodes.
1.3 Grow an overnight broth culture of E. coli (Done for you)
1. A single colony of K12 E. coli is lifted from a nutrient agar plate and added, using a
sterile toothpick, to a sterile test tube containing 10 mL Luria Broth (LB).
2. Culture is allowed to shake at 37°C overnight.
1.4 Transformation
Three hours before class the tech will take 1 mL of an ON culture and inoculate 50 mL of
fresh LB broth with it. It will shake at 37°C for two hours. This will produce cells in
exponential growth phase for you to make competent and transform.
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1. Divide broth culture into 2 sterile 30 mL screw top Oakridge centrifuge tubes; place
tubes in ice for 30 minutes.
2. Thaw out one tube of your plasmid DNA on ice.
3. Centrifuge the cultures at 4°C in the Beckman J2-21 for 10 minutes at 5000 rpm; decant
the supernatant into the collection flask provided.
4. Resuspend one pellet in 25 mL ice cold 50 mM CaCl2. Combine this resuspension
solution with the second bacterial pellet; place on ice for 20 minutes. Keep CaCl2 on ice
while waiting.
5. Centrifuge the cell suspension at 4°C in the Beckman J2-21 for 10 minutes at 5000 rpm.
6. Decant the supernatant and gently resuspend the pellet in 3 mL ice-cold 50 mM CaCl2;
place on ice for 5 minutes.
7. Dispense 2 aliquots of 0.3 mL cells in ice-cold 1.5 mL microfuge tubes; add 0.2 mL
of transformation buffer to each tube. Save remaining competent cells at 4°C.
8. Add 5 µL plasmid DNA to one tube. Add 5 µL TE buffer to the second tube. The second
tube will not receive plasmid DNA and will act as a control. Mix gently and leave on ice
for 20 minutes.
9. Heat shock cells for 1 min in 42°C water bath.
10. Plunge tubes into ice and let sit on ice for 5 minutes.
11. Add 0.7 mL LB to each tube and tap gently with finger.
12. Shake at 37°C for 60 minutes in Innova 4000.
**NOTE: during this hour incubation your TA or Lab Tech will demonstrate proper
spreading and streaking procedures for plating (See Appendix B and C). It is very
important that you understand sterile technique when working with bacteria so you
don’t contaminate your samples.
13. Plate 0.05, 0.1, and 0.3 mL of the cells with plasmid DNA onto LB amp plates. Use the
spread plate technique. Let the plates dry for 5 minutes right-side up before inverting
and placing in the incubator.
14. Streak (See Appendix H) (Do not use the spread plate technique) the contents of the
“no DNA tube” on an LB-AMP plate and an LB plate. The LB-AMP plate will act as a
negative control, while the LB plate will serve as a positive control.
15. Label plates appropriately with group number, date, type of bacteria, and any other
important information, such as how much bacteria was plated.
16. Incubate the plates at 37°C overnight (upside down); be sure to remove, wrap with
Parafilm and refrigerate the plates tomorrow!
Following Day: 1.5 Selecting for bacteria that carry the plasmid___
1. Examine transformed and no DNA control plates. (There should be no colonies on the
“No DNA” LB amp plate.)
2. Choose 3 well isolated colonies from the transformed plates. Streak (See Appendix H)
each colony on an LB-AMP plate.
3. Choose 2 well isolated colonies from the control (non-transformed) plate provided.
Streak each colony on one LB plate.
4. Incubate the plates overnight at 37°C.
5. Wrap the old plates in Parafilm and refrigerate.
14
1.6_Secondary selection of transformed bacteria_________________
1. Transfer 2 well-isolated colonies from 2 different transformed streaks and 2 control
colonies into separate 1 mL aliquots of sterile saline. Parafilm and refrigerate the old
plates.
2. For the transformed bacteria, streak (See Appendix H) 1 loopful of saline/bacteria
suspension onto an LB-AMP plate. Do this for each of the 2 samples.
3. For the control cells, streak 1 loopful of the saline/bacteria suspension onto an LB plate.
Be sure to label plates clearly!
4. Incubate at 37°C overnight; remove, parafilm and refrigerate for the next use.
5. Go to 1.16.
1.7 Preparing bacteria for the cracking gel (day before 1.8)
1. Using a marker, draw a line down the center of a new LB-AMP plate. Make a template
on paper with 1.5 cm x 1.5 cm squares on each half. Place the plate over the template.
2. Choose 2 LB-AMP plates from Day 1.6 that show the best growth with isolated colonies.
Using a loop, after sterilizing and briefly cooling it on the plate, select one colony from
the Day 1.6 plate and “fill in” the square on the agar on the plate. Repeat for the 2 nd
colony using the loop, again sterilizing it and cooling it before selecting a colony.
3. Repeat the procedure for the control, but use a fresh LB plate.
4. Incubate at 37°C overnight for at least 24 hrs, but less than 36 hrs.
5. Parafilm and refrigerate old plates.
1.8 Next day: Cracking gel
HAZARDOUS CHEMICAL INFO:
-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and
dispose of everything that has contacted EtBr in the appropriate solid waste container.
1. Prepare 0.7% agarose gel.
2. Use a sterile toothpick to scrape bacteria from the plates prepared the day before. Add
bacteria from each square to 250 µL of cracking buffer (one square for one tube of 250
µL of cracking buffer). Do this for transformed and non-transformed cells (you should
have a total of 2 microcentrifuge tubes). Vortex tubes to mix well.
3. Incubate the vortexed bacteria samples at 37°C in the hot water bath for 25 minutes.
4. Centrifuge for 15 minutes at 14,000 rpm.
5. Use a toothpick to remove the bacterial debris from the bottom of each tube. (You
won’t be able to see a pellet, but when you pull it out, it will look like a blue glob.)
6. Load the gel slowly and carefully:
Lane 1: 20 µL Hind III marker (Heat in 65C hot block for 7 minutes before loading)
Lane 2: 24 µL plasmid DNA solution (Use the same DNA plasmid and DNA loading buffer
dilution you used in 1.2)
Lane 3: Transformed supernatant
Lane 4: 50 µL cracking buffer only
Lane 5: Non-transformed supernatant
Note: Load as much transformed and non-transformed supernatant as possible (A wellformed well can hold ~50 L).
All empty lanes should again receive 20 µL 1X DNA loading buffer.
15
7. Run the gel for ~1 hour at 100 volts.
8. Stain with ethidium bromide, destain, and image. Look for genomic DNA, plasmid DNA
and RNA.
1.9 Labeling DNA with Biotin
-Salt-Saturated Phenol is to be used only wearing gloves under the hood. Dispose of all pipets
and liquid waste containing SS Phenol in appropriate disposals.
-Chloroform is to be used only wearing gloves under the hood. Dispose of all pipets and liquid
waste containing chloroform in appropriate disposals.
Part A: Labeling Reaction
1. Remove an aliquot of Plasmid DNA from the refrigerator and place on ice.
2. Add labeling reaction components to a 0.5 mL tube (on ice):
dH2O
128 µL
dNTP mix
28 µL
1X DNase I Buffer
19.9 µL
DNase I
0.1 µL*
Plasmid DNA
4µL
DNA Polymerase I
20µL
*Be EXTREMELY careful, this is a very small amount – try to see the DNase I in the tip or
ask the TA to check. You may gently rinse the tip in the reaction tube mixture to ensure
it has been added.
3.
4.
5.
6.
7.
Mix well and centrifuge for 5 seconds at desktop spinner.
Allocate 50 µL into four 0.5 mL microfuge tubes.
Incubate at 15°C for 2 hours in thermocycler.
Add 5 µL Stop Buffer to each tube and mix.
Incubate tubes at 65°C for 5 minutes in thermocycler.
Part B: Purification of DNA probes
1. Transfer liquid to consolidate solution from the four tubes into one 1.5 mL tube.
2. Add 4 µL 10% SDS to tube and mix.
3. Add 110 µL Chloroform and 110 µL SS Phenol to an empty 1.5 mL microcentrifuge tube.
4. Transfer DNA solution to chloroform phenol tube. Vortex 2 minutes and then centrifuge
for 2 minutes at 14,000 rpm.
5. Collect the top layer of liquid and transfer to a fresh 1.5 mL microcentrifuge tube.
Discard remaining liquid into waste container.
6. Add 220 µL chloroform to tube. Vortex 2 minutes and then centrifuge for 2 minutes at
14,000 rpm.
7. Collect top liquid layer and transfer to a clean tube. Discard remaining liquid into waste
container.
8. Add 40 µL 3 M Sodium Acetate (pH 4.8) and 800 µL cold 95% ethanol. Mix gently by
inverting tube.
9. Store at -20°C ON (at least 6 hours).
The Next Day:
10. Centrifuge for 5 minutes at 14,000 rpm.
16
11. Carefully remove the supernatant.
12. Resuspend the pellet in 1ml cold 70% ethanol. Centrifuge for 5 minutes at 14,000 rpm.
(1st Wash.)
13. Remove supernatant (ethanol).
14. Resuspend the pellet in 1 mL cold 70% ethanol. Centrifuge for 5 minutes at 14,000 rpm.
(2nd wash.)
15. Remove supernatant (ethanol). Let tube dry on lab bench for at least 1 hour.
16. Once dry, the TA will resuspend your probe in 12 µL TE buffer and store at -20° C.
1.10 Preparing for the Southern Blot (day before 1.11)
1. Using a marker, draw a line down the center of the underside of a fresh LB-AMP plate.
Draw two 1.5 cm x 1.5 cm squares on the underside of the plate, one on each half.
2. Using a sterile loop that has been cooled in the agar, pick one isolated colony from the
Day 1.6 LB amp transformed plate. “Fill in” one square on the fresh LB-AMP plate with
one colony. Repeat for the second square making sure to sterilize the loop and cool.
3. Repeat steps one and two, this time using an LB plate and the Day 1.6 non-transformed
cells.
4. Incubate both plates for at least 24 hours at 37°C.
1.11 Southern Blot
HAZARDOUS CHEMICAL INFO:
-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and
dispose of everything that has contacted EtBr in the appropriate solid waste container.
1. Run cracking gel exactly as you did on Day 1.8.
2. Stain with ethidium bromide, briefly destain, and examine the gel.
3. Photograph the gel before destaining completely—you will use this photograph later to
compare to the results of your southern blot.
4. Make sure to destain the gel for 5 minutes before continuing on to denaturing it.
5. Denature gel in 0.5 M NaOH/0.8 M NaCl for 30 minutes, rocking. Decant the solution
and repeat.
6. Rinse gel in dH2O for 1 minute.
7. Neutralize gel in 0.5 M Tris/1.5 M NaCl (pH 7.0) for 30 minutes, rocking. Decant the
solution and repeat.
8. While the gel is in the last stage of neutralizing, cut and hydrate the nitrocellulose filter
for 3 minutes in dH2O, then in 10X SSC until blot set-up is ready. Always wear gloves
when handling the nitrocellulose. Always handle the filter with forceps, and only
around the edges so as to not create blotches of background color.
9. While the gel is neutralizing, prepare the Test Spot.
a. Take your Biotin labeled probe out of the freezer and let thaw on ice.
b. Cut a piece of nitrocellulose approximately 1 cm x 1 cm. Make sure to cut
your test spot in a unique way so that you can identify it later. For
example you can cut one or two small notches on the side of the square
or cut off a corner.
c. Hydrate the test spot in dH2O for 3 minutes.
d. Soak the nitrocellulose in 10X SSC until the probe is thawed.
17
e. Remove the nitrocellulose from the SSC and place on a small Kimwipe™.
f. Add 2 µL of probe to the center of the square of nitrocellulose.
g. Let dry on Kimwipe™, then wrap in plastic wrap and store in the freezer
until 1.12.
10. Rinse the gel in 10X SSC for 3 minutes, rocking.
11. Assembling the Southern Blot (See Appendix J):
a. First the wick (a long strip of paper towel will work) needs to be placed on
the platform so that it can only touch the buffer on two sides.
b. Place three pieces of Whatman 3M filter paper on top of wick.
c. The gel should be placed on top of the filter paper and the nitrocellulose on
top of that. **Make sure the nitrocellulose and the gel are lined up in the
correct orientation so you can compare them later**
d. Place three more pieces of Whatman 3M filter paper on top of the
nitrocellulose.
e. Roll the filter paper with a test tube to remove any air bubbles.
f. A stack of cut paper towels at least 10 cm high should be assembled and
placed on top of the filter paper (all filter paper and paper towel should be
cut to the size of the gel).
g. Wrap the whole set up in plastic wrap to provide stability to the stack and
minimize evaporation.
h. Pressure should be applied to the top of the stack to enhance wicking
overnight.
(Your TA should demonstrate this and assist in the assembly)
12. Let Southern Blot transfer ON in 10X SSC.
1.12 Drying of Blot (Done for you)______________________________
1. Disassemble the Southern blot and rinse the nitrocellulose in 5X SSC for 2 minutes.
2. Dry on large Kimwipe™.
3. Bake nitrocellulose blot on a Kimwipe™ and test spot wrapped in plastic in vacuum oven
at 80°C for 2 hours.
4. Carefully place blot into hybridization bag and seal on all four sides.
5. Store blot and test spot in freezer.
1.13 Hybridization of the Southern Blot
1. Carefully unwrap your test spot.
2. Place your uniquely cut test spot in a small plastic tub containing all of the test spots
from the class. Your TA or lab tech will hydrate these in 2X SSC and then place them in
prehybridization solution and return them to you tomorrow for 1.14.
3. Cut a corner of your hybridization bag. Using a serological pipette, add 50 mL 2XSSC to
the bag to hydrate your blot. Try to remove all the air bubbles and reseal the corner of
the bag using the food sealer. The blot should be uniformly hydrated after several
minutes.
4. While the blot hydrates, denature 200 L of Herring sperm DNA (2 mg/mL) in 100°C hot
block for 10 minutes followed by plunging into ice water.
18
5. For prehybridization of the nitrocellulose blot, add the 200 µL of freshly denatured
Herring sperm to the prehybridization solution and mix.
6. Cut a corner of your bag and remove the 2XSSC by pouring it out. Using a serological
pipette, add the prehybridization solution (~8 mL). Reseal the corner after making sure
to push all of the air bubbles out of the bag. If there are still more air bubbles in the bag
after you have resealed the corner, set the bag upright and push all the bubbles to the
top of the bag. Reseal the bag across the top to trap the air bubbles away from the blot.
7. Incubate at 42°C while rocking for 2 hours. The volume of prehybridization solution
used should be 20 to 100 L per cm2 of the blot.
8. For hybridization, heat-denature 7 L of the probe made on 1.9 in a 0.5 mL microfuge
tube and 200 L of Herring sperm DNA in a different 0.5 mL microfuge tube by placing
the tubes in a 100°C hot block for 10 minutes. Before going into the hot block, wrap the
tops of the tubes with Parafilm so the writing is not removed in the next step.
9. Plunge the probe and Herring sperm samples into an ethanol ice slurry for fast chilling,
making sure not to erase all labels written in marker.
10. Just before use, add 5 µL of probe and 200 µL Herring sperm to the hybridization
solution.
11. Remove the prehybridization solution from the bag by cutting a corner and removing
the solution with a serological pipette. Add the hybridization solution to the bag using a
fresh serological pipette (20-100 L per cm2) and reseal using the same techniques
described in step 6. The blot should be hybridized at 42°C overnight while rocking to
achieve maximal sensitivity.
Following Day: 1.14 Washing and preparing the Southern blot
**All the washes in this section need to be completed while rocking.**
Decant and save the hybridization solution in an appropriate size tube. Store at 4C.
1. Wash the blot & test spot with 100 mL of 2X SSC/0.1% (w/v) SDS at room temperature
for 3 minutes. Decant the SSC and repeat.
2. Wash the blot & test spot with 100 mL of 0.2X SSC/0.1 % (w/v) SDS at room
temperature for 3 minutes. Decant the SSC and repeat.
3. Wash the blot & test spot in 100 mL of 0.16X SSC/0.1% (w/v) SDS at 50°C for 15 minutes.
Decant the SSC and repeat.
4. Rinse the blot & test spot in 100 mL of 2X SSC at room temperature for 1 minute.
5. Dry on large Kimwipe™ and then wrap in plastic wrap and store in refrigerator.
6. The hybridization mixture containing the biotin-labeled probe may be reused. Store the
mixture at 4C for several days or at -20C for longer periods. Placing the hybridization
solution in a boiling water bath and cooling on ice just prior to use should denature the
probe.
1.15 Development of Blot
HAZARDOUS CHEMICAL INFO:
-NBT/BCIP is highly toxic. WEAR GLOVES when handling and dispose of all liquid waste
containing NBT/BCIP in the appropriate waste container.
1. Wash the blot and small test square in Buffer 1 at room temperature for 1 minute with
sufficient buffer to cover. Decant Buffer 1 into the sink.
19
2. Incubate blot and test spot in Buffer 2 in a plastic container for 1 hour at 65°C, rocking,
with sufficient buffer to cover.
3. Decant off the Buffer 2, dry blots on Kimwipes™.
4. Dry blots at 80oC for 15 mins.
5. Wash the blots in freshly made streptavidin alkaline phosphatase (SA-AP) conjugate for
25 minutes at room temperature. 1 µL SA-AP per 1 mL Buffer 2.
(Add only enough SA-AP conjugate to cover the blots, ~10 mL. Use gentle agitation and
occasionally pipette SA-AP over the blots.)
6. Decant and save the SA-AP in a 15 mL tube. Save for step #9.
7. Wash the blot and test spot in Buffer 1 using 20 to 40-fold greater volume than
employed in step 1. Gently agitate blot for 15 minutes in Buffer 1.
(If you used 10 mL diluted SA-AP conjugate in step 1, wash with at least 200-400 mL
Buffer 1.) Decant Buffer 1 into the sink.
8. Wash the blots for 10 minutes in Buffer 3 while rocking. Decant Buffer 3 into sink.
*Do steps 9 and 10 at the same time and monitor the rate of color development. The
tube of saved SA-AP acts as a positive control.
** When you add the NBT/BCIP in the next steps, be sure to start a timer and note down
when color changes occurred.
9. Add 1 mL NBT/BCIP solution to the saved SA-AP. A blue color should develop overtime.
Wear gloves when working with NBT-BCIP.
10. Add 9 mL of NBT/BCIP solution to the blots. Allow the blots to develop for 15 minutes
to 1 hour. Agitate the Tupperware.
11. DNA bands will be most evident on only one side of the blot (check your notch for
correct orientation). Check your blot every 2 minutes to ensure that over-development
does not occur.
12. Once bands have developed, decant the NBT/BCIP solution in the appropriate waste
container and wash the blot in TE buffer. This will terminate the color development
reaction. The TE can then be decanted into the sink.
13. Let the blots dry on a large Kimwipe™. Once dry, wrap in plastic wrap and label. The lab
tech will image and distribute the blots to you for your notebooks.
14. Measure the image of the cracking gel and compare the relative position of the plasmid
band to the results of the blot. Interpret your results.
15. Record the amount of time development took in NBT/BCIP for the test and control
samples.
1.16 Designing Primers
Using the nucleotide sequence provided, you will design gene specific primers to use in
Polymerase chain reaction (PCR). In order to exponentially amplify a unit product that includes
all or part of the coding region for the glutathione S-transferase (GST) gene, you should design
two primers (Forward and Reverse). One primer should recognize part of the GST sequence
and the other primer should recognize part of the plasmid sequence. This will allow for the
amplification of the GST plasmid and in no way will endogenous GST be amplified. The forward
primer sequence can be taken directly from the 5’ 3’ nucleotide sequence provided; this
primer will bind, using Watson-Crick base pairing, to the 3’5’ complementary strand. (The
complementary strand is not shown with the sequence, but is included in the illustrated below.)
The forward primer will bind the 3’5’ strand in the 5’3’ orientation (see illustration). The
20
reverse primer needs to be the reverse compliment of the sequence provided, therefore
allowing for Watson-Crick base pairing to the 5’3’ sequence we give you, but binding at the 3’
end of the targeted sequence in the 5’3’ orientation (see illustration).
Please see the illustration below and go through the exercises to help you understand.
(Remember, DNA polymerase III, which we will use for PCR, must be primed and will produce a
complimentary strand in the 5’3’ direction.) Please make sure your final primers are written
in the 5’3’ orientation when you submit them.
Rules for Designing Primers
1. The primers should be between 18-25 nucleotides in length with a TM of
approximately 50-55oC.
2. TM= 2°C (A&T) + 4°C (G&C)
3. Primers should start and end with G’s or C’s
4. The nucleotide composition of the primer should be ~50% G/C and ~50% A/T.
5. Try to avoid long stretches of the same nucleotides (i.e. more than 4 of a particular
nucleotide in a row).
6. Make sure to avoid large amounts of complimentarity between the forward and
reverse primer and between the 5’ and 3’ ends of each primer.
Example of reverse primer design
1) 5’-GTCGTACGTACGGCGTCGTCC-3’ This is the sequence you want the reverse primer to
bind to.
2) 3’-CCTGCTGCGGCATGCATGCTG-5’ This is the sequence written backward; now it is
3’5’.
3) 5’-GGACGACGCCGTACGTACGAC-3’ This is the complementary bases of the sequence
above. This is the primer sequence, 5’3’ that will
bind to the sequence above and is the reverse
primer you would order from the company.
21
Now you try:
Here is a portion of the GST gene sequence that you want to use as a reverse primer
site. Design the primer that will bind to this sequence:
5’-GATATTAGATACGGTGTTTC-3’
Please draw on the DNA diagram below where your forward and reverse primers will bind and
which direction the Taq DNA polymerase will transcribe.
5’-------------------------------------------------------------------------------------------------------3’
3’-------------------------------------------------------------------------------------------------------5’
Part of the plasmid and the glutathione S-transferase (GST) nucleotide sequence (bold and
underlined) is shown below:
5’ – GTGGGGAAGGTGAGCGGATACAATTTCACACGGAAACAGTATTCATGTCCCCTATACTAG
GTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATC
TTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGG
CGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATT
GATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGAC
AAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCT
TGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAA
AGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAA
AATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAAC
CCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCC
AATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGC
TATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTT
GCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATC
TGGTTCCGCGTGGATCTCGTCGTGCATCTGTTGGATCCCCGGGAATTCATCGTGACTGACTGACGAT
CTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAG
CTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGT
CGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGGCGGAGTGTATAATTCTTGAAAAACAAAAGGG
CCTCGGGAAACGCCTATTTTTATAGGTTAATGGCATGAAAATAAATGGTTTTCTAAAACGTCAGGGGG
GCACTTTTTCGGGGAAAAGGTGGCGCGGAAACCCCTTATTTTGGTTTATTTTTTTTCTAAAAAAACATT
TTCAAAATAATGTTATCCCCCCCCTCATGGAAAAAAAAAATAAAACCCCCGGGGAAAAAAAAAGGGG
CTTTTTCAAAAATAAAAAAAAATATTTTGTAAAAAAAAAAAAAGGGGGGAGAGAA – 3’
22
Using the information we have given you, design primers to amplify all or part of the GST gene.
One or both of your primers should be from outside the GST sequence (bold and underlined),
meaning it is a part of the plasmid. Once you have designed the primers, fill out the
oligonucleotide request form. The primers will then be made on a DNA synthesizer.
The glutathione S-transferase protein consists of 232 amino acids. The sequence, using the
one-letter abbreviation for each amino acid, is shown below.
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYI
ADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDH
VTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK
SDLVPRGSPGIHRD*
Day Before 1.17
1.
Using the streaking method, plate fresh transformed and non-transformed bacteria
from your old plates (1.6).
1.17 PCR
1. Set up seven 0.5 mL PCR reaction tubes according to the PCR chart in Appendix K. Read
the chart carefully and make sure you add the correct amounts of reagent. PCR is a very
sensitive reaction and adding the incorrect amounts of reagents may cause poor results.
Appropriately label your tubes with your group color and tube number!
2. For Sample 4, pick up three individual colonies from your transformed plate with a
sterile toothpick and place into a 0.5 mL microfuge tube filled with 50 µL of sterile dH2O.
Do the same for Sample 5 using your non-transformed bacteria. Lyse the bacteria at
99°C for 5 min in the Thermocycler. Take 3 µL of this bacterial solution and use it as
your “template DNA”.
3. Before mixing the reactants, calculate how much water must be added to obtain a total
of 25 L in each reaction tube (including the Taq Polymerase). This is necessary because
the amount of template DNA that you add may differ from tube to tube.
4. Be sure each reagent is completely thawed before removing any. It is also a good idea
to mix the tube gently by flicking it and spinning it down in the bench top spinner.
5. If you add too much plasmid DNA, nonspecific amplification may occur—ask your TA
how much DNA to add based on the approximate concentration of your plasmid
samples.
6. Add all reactants, except the Taq, while the tubes are on ice.
7. Lastly add the Taq polymerase.
8. Once all reactants are added to the tubes, flick the tubes to mix them and spin them
briefly to bring all the liquid to the bottom of the tube. Note: Only spin tubes briefly (5
sec.), 0.5 mL tubes are thin-walled and can crack if microfuged for too long.
9. Keep the tubes on ice until the entire class is ready to load the thermocycler.
10. The thermocycler will run for approximately 3 hrs. After the 3 hour period is over, the
thermocycler will stay at a constant 4C until the tubes can be placed at -20°C by the lab
technician or TA. This will ensure that the PCR products do not degrade.
23
1.18 Examining the PCR product
HAZARDOUS CHEMICAL INFO:
-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and
dispose of everything that has contacted EtBr in the appropriate solid waste container.
1. Prepare a 2% agarose gel. Note: The 2% agarose solution will solidify quickly! Pour
gel while still relatively hot.
2. Remove 20 µL of PCR product from each tube; add to 4 µL of 6X DNA loading buffer.
Store the remaining PCR product at 4°C.
3. Once your gel is set, remove the comb and place the gel in the running box. Cover the
gel with 1X TAE buffer.
4. Load your DNA samples and 10 µL of 100bp ladder into the gel.
5. Add 20 µL of 1X DNA loading buffer to any empty lanes.
6. Run gel at 100 volts for 1.5 hours.
7. Once the electrophoresis is complete, stain your gel for ~20 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
8. Destain, examine, and photograph gel.
1.19 Searches of the sequence using BLAST
(Basic Local Alignment Search Tool)
The plasmid DNA has been sequenced. You will receive a printout of the results. You will
analyze this information using a computer program called BLAST.
To access the program, go to http://www.ncbi.nlm.nih.gov/BLAST
BLAST

Open the internet browser and go to http://www.ncbi.nlm.nih.gov/pubmed/

Scroll down the page and select BLAST from the popular searches menu

Select nucleotide BLAST and copy the entire GST nucleotide sequence (provided to you
via email) into the query sequence box

Make sure the database that is selected is nucleotide collection (nr/nt)

Click on the BLAST button at the bottom of the page and wait until the search is
completed (results will be presented in a new tab).
24
Module 2
RNA Interference
Introduction
In this module, a specific gene product of Paramecium tetraurelia is targeted and depleted
using an RNA Interference (RNAi) feeding method. Paramecia are fed with an RNAase IIIdeficient E. coli strain, HT115 bacteria, which have been transformed with a gene specific RNAi
construct or the empty RNAi vector. The RNAi construct, L4440 (see Appendix G) has opposing
T7 RNA polymerase promoters on either side of the cloned gene. When induced, these
promoters will allow for the expression of double stranded RNA. After 48 hours of feeding at
28°C, the cells are checked for behavioral changes. The level of target endogenous mRNA is
compared between test and control paramecia cells by total RNA extraction and semiquantitative Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR).
The lab tech has transformed E. coli HT115 cells. The “test” bacteria contain a plasmid with a
portion of the PAWN A gene, while the “control” bacteria contain the RNAi plasmid (L4440)
without the PAWN A gene. Both the test and control bacteria cells are resistant to ampicillin
and tetracycline.
2.1 Design Primers and PCR Protocol___________________________
641 bp – PAWN A Sequence
5’- ATGTATTTATTAATTTTAAGTATATTGTAATTTGGCATCGTGATTTAAGCTCAAGA
GACAAACAATACTGAAGAAGAGATTTCAGATTATTGTGATGCAGTTGCCAAAGCACTTC
TTTTAAGGTGAATGTAACAGTTTCAGATATTAACAATAAAAATTATTGTGTTGAAGGTGG
ATCTCGTGTGGCTTTATTCGACACAATTTAACAAGAAGATCAATATGTGTATTTGTCTGAC
ATTATTGTGCCAACTTATAACATTATCCAATTACTTGCGAACAATATTATCACGCTTCAGA
ATATGATAAAAAAGTAAGAATTTAAATACTATCAATTAGGCCAAAGCCAACTATA
GAAGCTCTATGAAGATTATAAGGCCACTGGAGTACCCAACTCTGATTGTTTGGGTATTGC
AAGATTCGTTTTCTGTGCTGAATAATTCAAATATTGCAGCACAGATGATGGAAATACCGA
TTATGAAATCTGCAGTTTCTTATGTGTCATTTGGCAAAATAGATGTCCTGATTACAGTGAT
ATTTACGATCGTGTTTGTGCTAATGGAGGAGGAGAAAATGGAAGATGCAGTTATGCAAT
TAACTATACTTTTCT GTTGTTTTTC ATTCTATTTT TATTATATTGA
An intron is located between nucleotides 313 to 333 (bold, larger font).
The sequence used for the RNAi construct is underlined. 560 bp (nucleotides 21  580).
25
Using this information, design primers that will allow us to look at the endogenous PawnA
sequence; primers that will not amplify exogenous material (the RNAi construct). Once you
have designed the forward and reverse primers, e-mail them in an excel spreadsheet to Dr.
Valentine. The primers will be ordered through the stockroom and be made on a DNA
synthesizer.
Based on the primers you designed, calculate the annealing temperature you would use. Fill out
the following tables with the PCR cycle information.
Temperature (°C)
Endogenous control PCR cycle conditions
Time (minutes)
Cycle
Initial Denaturation
Denaturation
Annealing
Extension
Final Elongation
2.2 Primer Optimization PCR
# of Cycles
1
1
____________________________
Set up the following reaction tubes to test the primers you have designed and ordered on
genomic Paramecium tetraurelia DNA (gDNA). Use the following table to set up your PCR
reaction tubes (0.5 mL thin-walled tubes).
You will first need to dilute your primers using dH20 to a stock concentration of 500 µM. Using
your stock tube, you should then prepare a working dilution of 20 µM, the final volume of your
working dilution should be 50 µL (Hint: C1V1 = C2V2).
For each primer set you will run four different reactions.
Components
Template DNA (~100 ng)
Tube 1
Tube 2
Tube 3
Tube 4
1 µL
1 µL
1 µL
1 µL
2.5 µL
2.5 µL
2.5 µL
2.5 µL
25 mM MgCl2
0 µL
1.5 µL
3 µL
4.5 µL
10 mM dNTPs
0.5 µL
0.5 µL
0.5 µL
0.5 µL
20 µM F primer
0.5 µL
0.5 µL
0.5 µL
0.5 µL
20 µM R primer
0.5 µL
0.5 µL
0.5 µL
0.5 µL
Taq Polymerase
0.25 µL
0.25 µL
0.25 µL
0.25 µL
dH2O
19.75 µL
18.25 µL
16.75 µL
15.25 µL
Total
25 µL
25 µL
25 µL
25 µL
10X Buffer
Based on the primers you received, determine what annealing temperature would be
appropriate to use (the calculated primer melting temperature (TM) and the actual TM are often
different). Also, what is the expected size of your PCR product?
26
2.3 Examine PCR product______________________________________
HAZARDOUS CHEMICAL INFO:
-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and
dispose of everything that has contacted EtBr in the appropriate solid waste container.
1. Prepare a 1.5% agarose gel using a 12 well comb. Note: The 1.5% agarose solution will
solidify quickly! Pour gel while still relatively hot.
2. Remove 15 µL of PCR product from each tube; add to 3 µL of 6X DNA loading buffer.
Store the remaining PCR product at 4°C.
3. Once your gel is set, remove the comb and place the gel in the running box. Cover the
gel with 1X TAE buffer.
4. Load your DNA samples and 10 µL of 100bp ladder into the gel.
5. Load 15 µL 1X DNA loading buffer to any empty lanes.
6. Run gel at 100 volts for ~1.5 hours.
7. Once the electrophoresis is complete, stain your gel for ~20 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
8. Destain, examine, and photograph gel.
2.4 Optimize PCR Protocol and practicing with paramecia using pulled pipettes _
Part A: Optimize your PCR if necessary.
1. After discussing the results with your instructors, optimize your PCR protocol.
Part B: Practice making and using pulled pipettes and counting Paramecium cells.
Next week, you will be required to count 350 paramecia to start your RNAi cultures and
observe the cells behavior in a barium chloride solution. In order to make the next week run
smoothly, it is important to learn to observe cells and transfer one cell at a time into the
behavior solution to observe backward swimming.
Before class: your TA will culture paramecia to practice with and will prepare hand-pulled
pipettes to make counting and transferring cells easier.
1.
2.
3.
4.
5.
Place 500 µL Resting buffer into the center depression of the depression slide.
Transfer 500 µL of paramecia culture to the first depression.
Observe the paramecia in the culture fluid and note their behavior in your lab notebook.
Learn to identify the anterior and posterior end of a cell.
Use your pulled pipette to transfer 10 cells to the resting solution. Note any reactions
or changes made by the cells in this new solution.
6. Continue to practice counting and transferring cells between depressions. At any point,
feel free to add new cells or dump out the depressions as you go.
7. The goals of this exercise:
a. Become accustomed with using the pulled pipettes
b. Observing cells and deciphering between the anterior and posterior of the cells
c. Transferring cells with as little accompanying fluid as possible
d. Counting cells efficiently
27
2.5 Agarose Gel of Optimized PCR Products________
_______
HAZARDOUS CHEMICAL INFO:
-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and
dispose of everything that has contacted EtBr in the appropriate solid waste container.
Part A:
1. Prepare a 1.5% agarose gel. Note: The 1.5% agarose solution will solidify quickly!
Pour gel while still relatively hot.
2. Remove 15 µL of PCR product from each tube; add to 3 µL of 6X DNA loading buffer.
Store the remaining PCR product at 4°C.
3. Once your gel is set, remove the comb and place the gel in the running box. Cover the
gel with 1X TAE buffer.
4. Load your DNA samples and 10 µL of 100bp ladder into the gel.
5. Add 15 µL of 1X DNA loading buffer to any empty lanes.
6. Run gel at 100 volts for ~1.5 hours.
7. Once the electrophoresis is complete, stain your gel for ~20 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
8. Destain, examine, and photograph gel.
Part B: Practice observing cells in barium chloride solution.
Next week, you will be required to observe cells in a barium chloride solution for backward
swimming. You must be able to transfer one cell at a time to this solution with very little
resting solution being transferred with the cell. You must also be able to decipher between the
anterior and posterior of the cell to determine if a Paramecium cell is swimming forward or
backward.
Before class: Your TA will culture paramecia to practice with and if necessary, pull more
pipettes. Your TA will discuss what is happening to the cells in the barium solution and what
types of reactions your might observe.
1. Place 500 µL Resting buffer into the center depression of the depression slide.
2. Transfer 500 µL of paramecia culture to the first depression.
3. Transfer ~20 cells with as little culture fluid as possible to the center well. If too much
culture fluid is transferred, add 500 µL Resting buffer to the last depression and transfer
the cells in the center depression to the last depression. Allow cells to sit for 10
minutes.
4. After 10 minutes, place 200 µL of 2 mM BaCl2 solution to the three wells of a second
depression slide. Label the slide that it contains the BaCl2.
5. Transfer one cell to one depression containing the BaCl2 solution and observe the cell in
that solution. Record the cells’ reaction in your notebook.
6. Transfer another cell to another depression and observe. Once all three depressions
have had cells added to them and the cells observed, wipe out the depressions with a
Kimwipe™ and add new 2 mM BaCl2.
7. Observe at least 20 cells.
28
2.6 ON Bacterial Culture (Done for you)_________________________
The lab tech will start ON cultures from freshly transformed test and control HT115 bacteria.
The control HT115 bacteria will have been transformed with the L4440 RNAi plasmid with no
insert. The test HT115 bacteria will have been transformed with the L4440 plasmid containing
a portion of the PAWN A gene. The bacteria cells will grow while shaking at 37C overnight in
LB containing 0.125 µg/mL tetracycline (TET) and 100 µg/mL ampicillin (AMP).
2.7 Induce Bacteria and Feed to Paramecium_____________________
Part A: Inducing HT115 bacteria
1. Five hours before lab, the lab tech will add 1 mL of the test and control HT115 bacteria
previously grown ON to two separate flasks containing 50 mL LB with 100 µg/mL AMP.
2. The bacteria will shake at 37C for approximately two hours.
3. After two hours of shaking, measure the OD value of the culture at 595 nm using the
spectrophotometer. LB-AMP will be used to blank the spec. Once the OD value reaches
between 0.3 and 0.4, proceed to the next step.
4. Add 125 µL 0.2 M IPTG to each 50 mL bacterial culture. Shake at 37°C for 3 hours.
Part B: Purging Paramecium Cells
(Your TA will demonstrate this procedure in MLS 224)
1. Obtain a 100 mL flask of paramecia grown in inoculated wheat culture from the 28C
incubator. (These cells were added to the inoculated wheat culture 4 days earlier and
are now feeling starved.)
2. Pour cells through funnel that has a small folded Kimwipe™ nestled inside into a 100 mL
pear-shaped centrifuge tube.
3. Balance the pear-shaped centrifuge tube against another pear-shaped centrifuge tube
filled with water. Centrifuge at ¾ speed for 2 minutes in the IEC HN-SII centrifuge (500 x
g).
4. Obtain a 15 mL plastic centrifuge tube containing 10 mL Dryl’s solution.
5. After centrifugation, your TA will demonstrate how to transfer the Paramecium cells
from the bottom of the pear-shaped tube to the Dryl’s solution using a glass Pasteur
pipette.
6. Keep the Paramecium cells in the Dryl’s solution until you are ready to count them.
Part C: Preparing the bacterial cultures for the Paramecium cells
1. After three hours of shaking, transfer the test and control bacterial cultures to large,
sterile centrifuge bottles and collect the bacteria by centrifuging at 5000 rpm, 4°C for 10
minutes in the Beckman J2-21 (JA-14 rotor).
2. Decant the supernatant into the waste jar.
3. Resuspend each bacterial pellet in 100 mL wheat culture containing 100 µL 100 mg/mL
AMP, 100 µL 8 mg/mL Stigmasterol, and 250 µL 0.2M IPTG. Transfer the resuspended
pellets back to their appropriate flasks.
4. Transfer 500 µL of paramecia in Dryl’s solution to a depression slide under a dissecting
microscope. Using your favorite pulled pipette, count and transfer 350 paramecia to
each flask that contain the test and control bacteria. Be sure not to allow the cells to dry
out. Storing them in a 1.5 mL microcentrifuge tube while you count the paramecia is OK.
5. Place flasks in 28C incubator. The starved paramecia will eat the bacteria.
29
2.8 Induce bacteria in the Paramecium culture (Done for you)_______________
After 24 hours of incubation, your TA will add 100 µL 100 mg/mL ampicillin and 250 µL 0.2M
IPTG to each of the cultures and continue to incubate the flasks at 28C.
2.9 Harvest cells and Isolate RNA
HAZARDOUS CHEMICAL INFO:
β-Mercaptoethanol is toxic. WEAR GLOVES when handling, and dispose of all solid waste
(pipets, gloves, or anything else that has come in contact with the 2-Mercaptoethanol) in the
appropriate waste container. Do not inhale fumes.
Induce bacteria in the Paramecium culture (Done for you)
After 48 hours of incubation, your TA will add 100 µL 100 mg/mL AMP and 250 µL 50 mM IPTG
to each of the cultures and continue to incubate at 28C.
Part A: Checking Paramecium Phenotypes
The TA will describe the expected phenotypes of the Test and Control paramecia. View the
cells in a depression slide under a dissecting microscope to confirm the expected phenotypes of
both treatments.
1. Take 500 µL of Test and Control cultures and transfer to a depression slide. Mark the
depression slides as Test and Control.
2. Start with one cell type and transfer ~30 cells to 500 µL of Resting Solution in another
depression slide. Try not to transfer much culture medium.
3. Wait 10 minutes.
4. Place 200 µL of 2 mM BaCl2 solution to another depression slide.
5. After 10 minutes, set up the second cell type by transferring ~30 cells to 500 µL resting
buffer in a well labeled depression slide. Cover with a cover slip and set aside.
6. From the original cell type that has been in the resting buffer for 10 minutes, transfer 1
cell at a time from the resting solution to the 2 mM BaCl2 solution. Note the swimming
behavior of each cell. Change the 2 mM BaCl2 solution often (every one or two cells).
 A cell that swims backward should be scored as a “1.”
 A cell that does not swim backward should be scored as a “2.”
7. Now repeat with the second cell type.
8. Check at least 15 cells of each type, test and control.
Part B: Filtering and Washing the Paramecium cells
(Your TA will demonstrate procedure in MLS 224)
1. Filter paramecia through folded small Kimwipes™ into 100 mL pear-shaped centrifuge
tubes. Label as Test and Control.
2. Balance pear-shaped tubes and centrifuge at ¾ speed for 2 minutes in the IEC HN-SII
centrifuge. Use Dryl’s solution to balance the tubes.
3. Using a glass Pasteur pipette, remove the cells collected at the bottom of the tube.
(Your TA will demonstrate how to use circular motion to aid in removing a concentrated
30
amount of cells.) Dump the supernatant into the waste container provided and then
place the pipette containing the cells back into the empty pear-shaped centrifuge tube.
4. Add approximately 100 mL Dryl’s solution to the pear-shaped centrifuge tube containing
the cells to wash them. Use the Pasteur pipette to gently mix the cells, be sure to get
any that remain in the stem of the tube.
5. Balance and centrifuge the pear-shaped tubes at ¾ speed for 2 minutes in the IEC HN-SII
centrifuge.
6. Again using a sterile Pasteur pipette, remove the cells with as little culture fluid as
possible and transfer the paramecia cells to separate 1.5 mL microcentrifuge tubes.
Continue to next section and start RNA Prep within a few minutes of collecting cells.
Part C: RNA Prep (To be completed in MLS 007A by students)
1. Disinfect lab bench, pipettes, Sharpie, and gloves of all RNases. Spray surfaces with
“RNase Away” and then wipe with paper towel. It is crucial that the following protocol
be performed in an RNase-free environment. Your TA will explain the importance of
RNase-free conditions.
2. Centrifuge cells for 10 seconds in the bench-top spinner and remove the majority of the
supernatant using a P-1000 pipette. Approximately 100 µL should remain.
3. Add 350 L of the lysis buffer (RA1) to each of the 1.5 mL tubes of cells.
4. Next, add 3.5 L of β-Mercaptoethanol to each tube. Use β-Mercaptoethanol in the
hood and place all pipette tips that come into contact with β-Mercaptoethanol into
designated waste container!
5. Use 1 mL syringes with an 18G or 22G needle to lyse each cell type by passing it through
the syringe three times. Careful not to expel the solution too quickly, it can spurt out of
the microfuge tube.
6. Using two collection tubes, place a pink filter column inside each collecting tube and
pipette the test and control cell solutions onto the center of the pink filter. Label both
the collecting tube and the filter.
7. Centrifuge the tubes for 1 minute at 12,000 rpm in the microfuge.
8. Remove the filter and discard, saving the collection tubes with the supernatant.
9. Add 350 L of 70% ethanol to the samples in the collecting tubes. Pipette up and down
to mix.
10. Set up a blue spin column for each sample with a collecting tube and label well (both the
collecting tube and the spin column).
11. Transfer each sample to a blue spin column and centrifuge for 1 minute at 8,000 rpm.
12. Place each blue spin column into a new collecting tube and label. Discard the
supernatant and the used collecting tube.
13. Add 350 L Membrane Desalting Buffer (MDB) and centrifuge at 12,000 rpm for 1 min.
14. Per sample, mix 90 L of DNase Reaction Buffer and 10 L of DNase I stock solution in a
fresh 0.5 mL microcentrifuge tube.
15. Add 95 L of the DNase reaction mixture to the center of each blue spin column. Do not
contact column and make sure DNase is absorbed. Allow the columns to sit at room
temperature for 30 minutes.
16. After 30 minutes, add 200 L RA2 Buffer to the spin columns and centrifuge for 1
minute at 8,000 rpm. (If the tip of the column outlet comes into contact with the flow31
through for any reason, discard the flow-through and centrifuge again at 12,000 rpm for
1 minute.)
17. Place spin columns into new collecting tubes.
18. Add 600 L of RA3 Buffer to the spin columns and centrifuge for 1 minute at 8,000 rpm.
19. Discard the flow-through and place the columns back into the collecting tubes.
20. Add 250 L RA3 Buffer to the spin columns and centrifuge for 2 minutes at 12,000 rpm.
21. Place the spin columns into supplied RNase-free 1.5 mL microcentrifuge tubes and label.
22. Elute the RNA by adding 20 L of RNase-free water directly into the center of the spin
column. Allow water to enter the column (wait 1 minute) and centrifuge for 1 minute at
12,000 rpm.
23. Remove the liquid from the collecting tube and place it in a clean 0.5 mL centrifuge tube
and keep on ice. Add a second 20 µL of RNase-free water to the spin column, allow the
water to seep in (wait 1 minute) and centrifuge for 1 minute at 12,000 rpm.
24. Discard spin column, combine to two 20 µL eluates. Remove 3 µL and place in a fresh
0.5 mL tube for nanodrop.
25. Place all the RNA samples in the -80C freezer.
2.10 Making cDNA
__________
Your TA will determine the total RNA yield for each of your samples using the Nanodrop. The
concentration will be reported in ng/L.
1. Calculate the volume of RNA needed to equal 5 µg per tube using the RNA
concentration provided by the TA.
a. Note: The concentration of the test and control sample MUST be equal here.
b. Your volume cannot exceed 11 µL of RNA and water; typically one of the samples
will limit the concentration of RNA you will start with. You can use less than 5 µg.
2. Disinfect lab bench, pipettes, Sharpie, and gloves of all RNases. Spray surfaces with
“RNase Away” and then wipe with paper towel. It is crucial that the following protocol
be performed in an RNase-free environment.
3. Set up the RT-PCR using 0.5 mL tubes and the following table. Tube 1 will contain test
RNA and Tube 2 will contain control RNA (positive control). Tube 3 is the negative
control which will contain one type of RNA and no SuperScript III.
Reagents (µL)
Tube 1 (Test)
Tube 2 (+ Control) Tube 3 (- Control)
RNA * 5 µg
dH20 *
50 µM Oligo dT
1 µL
1 µL
1 µL
10 mM dNTPs
1 µL
1 µL
1 µL
Total volume
13 µL
13 µL
13 µL
*The remaining volume of each tube should be made up of water (total volume = 13 µL). Be
sure to add the water and the RNA first, as these will be larger volumes.
4. Incubate tubes at 65C for 5 minutes then put on ice and add:
32
Reagents (µL)
5X First-Strand Buffer
0.1 M DTT
RNase Out
SuperScript III
Water
Total Volume
Tube 1 (Test)
4 µL
1 µL
1 µL
1 µL
0
20 µL
Tube 2 (+ Control)
4 µL
1 µL
1 µL
1 µL
0
20 µL
Tube 3 (- Control)
4 µL
1 µL
1 µL
0
1 µL
20 µL
5. Place tubes in thermocycler and set to the following conditions:
cDNA synthesis Conditions
30 min
50C
30 min
55C
15 min
70C
6. Remove tubes from thermocycler and place at -20C.
2.11 Template Control and Endogenous PCR
_____
Part A: Serial Dilutions
1. Make serial dilutions of both the Test and +Control cDNA according to the following
chart:
Serial Dilution
1:10
1:50
1:100
1:500
cDNA
2 µL of cDNA
2 µL of previous dilution
4 µL of previous dilution
2 µL of previous dilution
H2O
18 µL
8 µL
4 µL
8 µL
2. Keep tubes on ice while preparing PCR tubes.
Part B: Template Control PCR
Calmodulin is a protein highly expressed in Paramecium. We will detect the mRNA levels of the
calmodulin gene in both the Test and +Control samples to determine if there are equal
concentrations of mRNA in both the Test and the +Control. We will also use Calmodulin
primers on the –Control cDNA.
Calmodulin primers:
Forward: 5’ CTGAAGCTGAACTTCAAG 3’
Reverse: 5’ TCATTTAGAAACCATCATTCT 3’
Product length: 330-350 bp
33
1. Set up and label nine 0.5 mL tubes according to the table below (UD=undiluted).
Calmodulin PCR (Note: the endogenous set up is different)
Tube Treatment
µL of cDNA
µL of Master Mix
1
UD Test
1
49
2
1:10 Test
1
49
3
1:100 Test
1
49
4
1:500 Test
1
49
5
UD +Control
1
49
6
1:10 +Control
1
49
7
1:100 +Control
1
49
8
1:500 +Control
1
49
9
UD -Control
1
49
2. Each tube will contain 1 µL of the respective cDNA dilution and 49 µL of master mix. The
master mix contains the reagents necessary for the PCR. Make the master mix in a 1.5 mL
tube by adding:
1 rxn
10 rxns
Sterile dH2O
37.5 µL
375 µL
10X PCR buffer
5 µL
50 µL
25mM MgCl2
3 µL
30 µL
10 mM dNTPs
1 µL
10µL
20 µM Calmodulin F primer 1 µL
10 µL
20 µM Calmodulin R primer 1 µL
10 µL
Taq Polymerase
0.5 µL
5 µL
49 µL
3. Mix the solution well by pipetting up and down. If you flick the tube to mix, be sure to
spin down in the bench-top spinner.
4. Add 49 µL of master mix to each labeled 0.5 mL tube.
5. Add 1 µL of respective cDNA dilution to each tube. Flick each tube to mix well.
6. Spin tubes in table top spinner for 10 seconds.
7. Place tubes in the thermocycler making sure the caps are completely closed and the
contents of the tube are at the bottom of each tube and not stuck up on the sides or in
the cap.
8. Run PCR with the following conditions:
Calmodulin PCR Conditions
5 min
Initial Denature
95C
1 min
95C
24X
1 min
42C
40 sec
72C
10 min
Final Elongation
72C
HOLD
****************
4C
34
Part C: Endogenous gene PCR
The endogenous PCR will allow us to check the mRNA levels and see if the PAWN A gene
expression was down regulated from to RNA interference in the test cells compared to the
controls.
1. Set up and label nine 0.5 mL tubes according to the table below.
Endogenous PCR
Tube Treatment
1
UD Test
2
1:10 Test
3
1:50 Test
4
1:100 Test
5
UD +Control
6
1:10 +Control
7
1:50 +Control
8
1:100 +Control
9
UD -Control
µL of cDNA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
µL of Master Mix
24.5
24.5
24.5
24.5
24.5
24.5
24.5
24.5
24.5
2. Each tube will contain 0.5 µL of the respective cDNA dilution and 24.5 µl of master mix.
The master mix contains the reagents necessary for the PCR. Make the master mix in a
1.5 mL tube by adding:
*Sterile dH2O
10X PCR buffer
*25mM MgCl2
10 mM dNTPs
20 µM Endogenous F primer
20 µM Endogenous R primer
Taq Polymerase
1 rxn
_____ µL
2.5 µL
_____ µL
0.5 µL
0.5 µL
0.5 µL
0.25 µL
24.5 µL
10 rxns
_______ µL
25 µL
_____ µL
5 µL
5 µL
5 µL
2.5 µL
245 µL
* These amounts will vary based on your results from 2.2 and 2.4.
3. Mix the solution well by pipetting up and down. If you flick the tube to mix, be sure to
spin it down briefly in the bench-top spinner.
4. Add 24.5 µL of master mix to each 0.5 mL labeled tube.
5. Add 0.5 µL of respective cDNA dilution to each tube. Flick each tube to mix well.
6. Briefly (10 sec) spin each tube down in the bench-top spinner.
7. Place tubes in the thermocycler, making sure the caps are completely closed and the
contents of the tube are at the bottom of each tube and not stuck up on the sides or in
the cap.
8. Run PCR with the conditions you determined during 2.2 and 2.4:
35
Endogenous PCR Conditions
Temp
Time (min)
Cycle
Initial Denature
30X
HOLD
Final Elongation
****************
9. When the PCR is complete your TA will remove the PCR product tubes from the
thermocycler and place at 4C.
2.12 Template Control (Calmodulin PCR) Gel
HAZARDOUS CHEMICAL INFO:
-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and
dispose of everything that has contacted EtBr in the appropriate solid waste container.
1. Prepare a 2% agarose gel with a 12-well comb. Note: The 2% agarose solution will
solidify quickly! Pour gel while still relatively hot.
2. Remove 10 µL of PCR product from each tube; add 2 µL of 6X DNA loading buffer. Store
the remaining PCR product at 4°C.
3. Once your gel is set, remove the comb and place the gel in the running box.
4. Cover the gel with 1X TAE buffer and load your samples.
5. Load 10 µL of 100 bp ladder to a lane. Be sure to note down what sample was loaded
into what lane. Any empty lanes should receive 10 µL of 1X DNA loading buffer.
6. Run gel at 120 volts for ~1 hour.
7. Once the electrophoresis is complete, stain your gel for ~20 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
8. Destain, examine, and photograph your gel.
2.13 Endogenous mRNA Gel
HAZARDOUS CHEMICAL INFO:
-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and
dispose of everything that has contacted EtBr in the appropriate solid waste container.
1. Prepare a 1.5% agarose gel with a 12-well comb. Note: The 1.5% agarose solution will
solidify quickly! Pour gel while still relatively hot.
2. Remove 10 µL of PCR product from each tube; add 2 µL of 6X DNA loading buffer. Store
the remaining PCR product at 4°C.
3. Once your gel is set, remove the comb and place the gel in the running box.
4. Cover the gel with 1X TAE buffer and load your samples.
5. Load 10 µL of 100 bp ladder to a lane. Be sure to note down what sample was loaded
into what lane. Any empty lanes should receive 10 µL of 1X DNA loading buffer.
6. Run gel at 120 volts for 45 minutes to 1 hour.
7. Once the electrophoresis is complete, stain your gel for ~20 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
36
8. Destain, examine, and photograph your gel.
2.14 Analyze Data_____________________________________________
Work with your partner and discuss the results. Your TA will be there to answer any questions
you may have.
37
Module 3
Proteomics
Introduction
In this module, you will compare the proteins found in wild type and mutant Paramecium ciliary
membranes. You will run a one dimensional polyacrylamide gel, remove and cut into bands the
lanes from the mutant and wild type protein samples, then compare the proteins present in
those samples using mass spectrometry.
3.0 Isolation of Cilia (Done for you)
_____
Your TA will prepare the cilia from Paramecium using the following protocol. Approximately 6 L
of wild type cells and 9-12 L of mutant cells are needed. The cells are not all harvested on the
same day. Below is the protocol for harvesting the Paramecium cells, typically 3 L at a time (All
solution recipes are shown in Appendix A):
1. Filter Paramecium cell culture by pouring culture through a funnel lined with cheese
cloth sandwiched between 2 large Kimwipes™ into a clean 2800 mL flask.
2. Pour the culture through and IEC Clinical continuous flow centrifuge to concentrate the
cells into ~300 mL.
3. Concentrate the cells further using an IEC HN-SII clinical centrifuge and pear-shaped
tubes. Cells are spun for 2 minutes at ¾ speed (~500 x g) and collected using a Pasteur
pipette.
4. Pipette cells into a beaker containing 200 mL of room temperature Dryl’s solution.
Centrifuge again in pear-shaped tubed for 2 minutes at ¾ speed. Remove trichocysts
(fluffy layer on top of pellet) and put in waste container. Transfer cells to 200 mL fresh
Dryl’s solution. Mix and centrifuge again. The cells are washed a total of 3 times.
5. After the final wash, add cells to a flask containing 40 mL cold Dryl’s solution, add 40 mL
cold STEN buffer. Keep on ice for 10 minutes to immobilize the cells.
6. Add 16 mL Cilia Shock Buffer to cells, swirl the beaker and place on ice for 2-5 minutes.
During this time the cells will deciliate – examine under inverted microscope to watch
the progress of deciliation and to make sure that the cell lysis is minimal (preferable
<1%). If cells begin to lyse, spin down immediately.
7. Transfer cell solution to pear-shaped flasks and centrifuge for 2 minutes at full speed
(~850 x g). Pour the supernatant into 2 clean pear-shaped tubes, leaving cell bodies in
the old tubes. Spin supernatant again for 2 minutes at full speed.
8. Transfer supernatant to 30 mL Corex tubes with rubber sleeves. Spin in Beckman J2-21
Centrifuge (JA-17 rotor) at 14,500 rpm for 20 minutes.
9. Add 3 mL of 10 mM Tris EDTA pH 8.3 to one tube and vortex for 5 minutes. Transfer the
resuspension to the next tube and vortex for 5 minutes. Continue to consolidate the
pellets until all pellets have been resuspened.
10. Rinse the empty tubes with 2 ml Tris-EDTA pH 8.3 to collect any residual cilia. The total
volume of the sample should now be ~5 mL.
11. Place the resuspended cilia in a fresh 15 mL Corex tube and wash the last tube with 1-2
mL Tris EDTA pH 8.3.
12. Spin at 19,500 rpm in JA-20 rotor for 30 minutes.
13. Pour off the supernatant. Resuspend the pellet in 400 - 600 µL of 10 mM Tris (pH 8.0).
Be sure the suspension is homogenous. Store at -80°C for future use.
38
3.1 Ultracentrifugation_________________________________________
Each group will be provided with a cilia protein sample. Three groups will be given wild type
samples and three groups will be given eccentric, XntA1 (mutant) samples. Keep these on ice.
1. You are provided 66% sucrose. In three 15 mL tubes, create three concentrations of
sucrose (55%, 45% and 20%) using the calculations in Appendix L.
2. Observe the TA making a step gradient of sucrose. Label your ultracentrifuge tube with
your group color.
3. Now, create the sucrose gradient by first adding 0.7 mL of 66% sucrose to the Ultraclear
Ultracentrifuge tube, then 1.4 mL 55% sucrose, then 1.4 mL 45% sucrose and finally 0.7
mL 20% sucrose.
4. Carefully place 200 to 300 µL of your sample on top of the sucrose gradient. Rinse the
empty protein sample tube with 100 µL of 10 mM Tris (pH 8.0) + protease inhibitors and
add to the gradient. Make sure your Ultracentrifuge tube is labeled: Wild type (control)
or XntA1 (mutant).
5. Balance the ultracentrifuge tubes in their buckets with 10 mM Tris (pH 8.0) + protease
inhibitors. Note the number on the bucket that corresponds to your sample.
6. Your TA will set up and run the ultracentrifuge.
7. The Beckman L8-80M Ultracentrifuge (Sw60Ti rotor) will spin at 45,000 rpm at 4°C for
1.5 hours.
8. After centrifugation, three sample interfaces should be visible (see Appendix M). We
are only interested in collecting the pure ciliary membrane which is between the 20%
and 45% sucrose layers.
9. Use an insulin syringe to poke a hole in the side of the ultracentrifuge tube and suck out
the white, cloudy interface between the 20% and 45% sucrose layers. Place the
collected sample into a fresh 15 mL Corex tube.
10. Add 12 mL of 10 mM Tris pH 8.0 + protease inhibitors to each tube. Cover the tube with
parafilm and invert numerous times to mix well.
11. Balance the tubes in their rubber sleeves. Make sure the tubes are labeled with your
type of sample and group.
12. Centrifuge in the Beckman J2-21 (JA- 17 rotor) at 17,000 rpm at 4°C for 30 minutes.
13. Immediately remove the samples, pour off the supernatant and discard in the waste
container. Allow tubes to rest upside down on a Kimwipe™ for a minute and then place
on ice.
14. Resuspend the pellet in 50 µL of Membrane Buffer with protease inhibitors. While
resuspending the pellet, try to keep the tube on ice.
15. Move the sample to a 0.5 mL tube that is well labeled. Rinse the Corex tube with an
additional 25 – 50 µL of membrane buffer + protease inhibitors to remove all the sample
and combine this with your original sample. Mix well.
16. Remove 16 µL from your sample. Again, make sure the sample was well mixed. Place
the 16 µL in a labeled microfuge tube for the Pierce BCA Protein Assay.
17. Store both tubes (samples) at -80°C.
39
3.2 Preparation of Resolving Gel
HAZARDOUS CHEMICAL INFO:
-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste
(pipets, gloves, or anything else that has come in contact with the acrylamide) in the
appropriate waste container.
-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets,
gloves, or anything else that has come in contact with the TEMED) in the appropriate waste
container. Do not inhale fumes.
-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling,
and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the
TEMED) in the appropriate waste container.
2.
3.
4.
5.
Obtain one 1.5mm spacer glass plate and one short glass plate; wipe with methanol
and a Kimwipe™ until you hear a “squeaky” noise. Handle glass plates at edges.
Wear gloves!
Assemble the gel casting apparatus (see Appendix N). Assemble on a flat surface
and then clamp shut.
Insert the 1.5mm comb and use a Sharpie to draw a line across the glass 0.7 cm
below the bottom of the wells of the comb. Once the line is drawn, remove the
comb.
Test to see if the apparatus is leak-proof by squirting some water in between the
glass plates and look for leaks. If leaks occur, a tighter seal must be achieved. Make
sure to remove the water before pouring the gel. Use a piece of Whatman filter
paper to remove water droplets from between the plates of glass.
Before you pour your gel, have the TA or lab tech check your apparatus!
6. Prepare a 12% resolving gel in a 15 mL tube according to the following directions.
DO NOT ADD THE FRESHLY MADE AMMONIUM PERSULFATE UNTIL YOU ARE READY
TO POUR THE GEL.
Sterile dH2O
4X Resolving Buffer pH 8.9
30% Acrylamide stock
TEMED
Last:
Fresh 10% Ammonium persulfate
3.29 mL
2.60 mL
4.00 mL
10 L
100 L
7.
8.
Gently invert the solution to mix WELL.
Using a serological pipet, quickly pipette the acrylamide mixture into the cassette by
allowing the solution to run down along the side of the spacer. Add the acrylamide
solution until it is just barely above your Sharpie line. Try to avoid making bubbles.
9. Overlay the acrylamide immediately with dH2O using a P-1000. Do this by gently
adding the dH2O. You will be able to see a distinct line between the dH2O and the
resolving gel solution.
10. Allow the gel to polymerize for AT LEAST 30 minutes. Any extra acrylamide mix in
your tube will be a good gauge for polymerization. (Make sure the cap is on.)
40
11. After your gel has polymerized, remove the water and check the interface – it MUST
be flat. If it is not, the gel must be poured again.
12. Once your gel has polymerized and has a flat interface, cover completely with water
and wrap the gel/casting apparatus in a damp paper towel and then plastic wrap.
Label appropriately. Store in the cold room.
3.3 Pierce BCA Protein Assay
Pierce BCA Protein Assay (See Appendix O)
Use the chart provided to develop a standard curve using BSA standards and to determine
protein concentration.
1. Dilute protein sample: Make two 1:50 and 1:20 dilutions of your protein sample. For
example, to make the 1:20 dilution, add 5 L of your sample to 95 L of sterile dH2O.
For the 1:50 dilution add 2 L of your sample to 98 L of sterile dH2O. The dilutions
should be done twice, not one large dilution that you split in half.
2. Add BSA and dH2O according to the directions in Appendix O. These should also be done
in duplicate. This set up will provide two identical reactions.
3. Make dye solution only once all your standards and unknowns are prepared: Use
Solutions A and B from the Pierce Protein Assay Kit; Solutions A and B should be mixed
50:1. Show the amount of dye you plan to make to the TA or technician. Be sure to plan
on a few extra tubes.
4. Add 2 mL of the dye to each one of your samples and standards.
5. Incubate at 37°C for 30 minutes.
6. Get OD values for standards and samples: TA will assist in the operation of the spec.
Obtain OD values for one set of standards and unknowns, then repeat the process for
the second set of standards and unknowns.
a. Place your standard into a clean cuvette to blank the spec.
To clean the cuvette, rinse with dH2O. Make sure to dry the outside of the
cuvette with a Kimwipe™. Handle the cuvette only on the frosted sides.
b. Read OD at 562 nm.
c. Obtain readings for the first set of standards and unknowns.
d. Re-blank the spec with the second blank for the second set of standards and
unknowns. Obtain the OD’s for the second set.
If only using 1 cuvette, make sure to rinse with dH2O between each standard.
7. Find the average of each standard and sample duplicates.
8. Establish a standard curve using the OD values obtained with your BSA standards:
graph Concentration (x-axis) vs. OD (y-axis) on excel. Using this graph and the line
equation, calculate the protein concentrations in your two samples.
Do not force the line through 0.
See Appendix Q for assistance.
9. E-mail your graph and calculated unknown concentrations to Dr. Valentine
(Megan.Valentine@uvm.edu). Be sure to put the graph, OD values, and calculated
concentrations in your lab notebook.
10. You will need your calculated concentrations of your protein sample for the next class.
41
3.4 SDS-PAGE
HAZARDOUS CHEMICAL INFO:
-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste
(pipets, gloves, or anything else that has come in contact with the acrylamide) in the
appropriate waste container.
-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets,
gloves, or anything else that has come in contact with the TEMED) in the appropriate waste
container. Do not inhale fumes.
-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling,
and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the
APS) in the appropriate waste container.
-Coomassie Blue Stain and Coomassie Blue Destain are highly flammable and irritating to the
skin. WEAR GLOVES when handling and dispose of in appropriate waste container.
1. Remove the resolving gel from the cold room and pour a 4% stacking gel.
2. Make sure you have cleaned your comb with ethanol.
3. Mix the following components in a 15 mL tube.
Sterile dH2O
4X Stacking Buffer pH 6.8
30% Acrylamide stock
TEMED
6.10 mL
2.50 mL
1.30 mL
10 L
Fresh 10% Ammonium persulfate
50 L
Last:
4. Before adding the ammonium persulfate, pour the dH2O off the resolving gel and
remove any residual water using a piece of Whatman filter paper.
5. Add the ammonium persulfate to your tube. Mix gently.
6. Pour the stacking gel as you did the resolving gel all the way to the top of the small glass
plate using a serological pipet. Take caution when inserting the comb into the gel, it may
spurt out. If the acrylamide mix overflows when inserting the comb, it’s okay. Make sure
there are no air bubbles in between the wells or at the bottom of the teeth of the comb.
This is VERY IMPORTANT; the stacking gel will not polymerize if the comb is not
clean, or if there is air between the wells. Ask your lab tech to double check your
set up.
7. Allow 30 minutes for the gel to polymerize. Thaw your protein samples on ice while
waiting.
8. Once the gels are set, remove them from the casting stand and assemble in the gel box
(See Appendix P). Note: Do not remove the comb yet.
9. Add 1X PAGE Running buffer to the upper chamber. The buffer level should be half way
between the top of the big and small glass plates.
10. Add 1X PAGE Running buffer to the lower chamber until the appropriate level for the
number of gels in the box is reached.
11. Carefully remove the comb.
42
Gel Set Up
Your TA will help you determine how much sample you are going to load based on the
concentration of your protein sample. Groups will share proteins samples between control
(wild type) and mutant (XntA1). Each gel will have equal concentrations of the control and
mutant pure ciliary membrane samples and a prestained protein marker (see Appendix I). Your
TA will help you to equalize the concentrations of control and mutant samples; these must be
approximately equal in order for the samples to be comparable. Any empty lanes should be
loaded with 1X SDS sample buffer.
1. Calculate the volumes of wild type and mutant sample, 10X SDS sample buffer, and
Membrane buffer needed for each tube:
a. Before using the 10X SDS Sample Buffer, add 50 µL of β-ME to 500 µL of 10X SDS
Sample Buffer.
Be sure to do this in the fume hood.
b. Calculate the volume of your protein that provides *60 µg. The remaining
volume should be made up with 10X SDS sample buffer and membrane buffer
so the final volume should be 50 µL:
*60 µg protein
_____________ µL
Membrane Buffer
_____________ µL
10X SDS sample Buffer
5 µL
2.
3.
4.
5.
6.
7.
8.
c. Obtain the other sample (wild type or mutant) from another group – we will help
match you all up. Double check the other group’s concentration calculation so
we are sure everything is correct.
*We reserve the right to alter concentrations or final volumes based on the
concentrations of proteins obtained by the class. If a large amount of protein is
obtained, we may increase the concentrations to 100 µg. If not enough protein is
collected, we may decrease the concentration and/or loading volume.
Add the calculated volumes of membrane buffer, protein, and prepared 10X SDS sample
buffer to a 1.5 mL tube. Place ANY remaining protein samples in the freezer.
Make sure your samples are well labeled.
Boil samples for 5 minutes right before you are ready to load. After boiling, keep
samples on ice while loading gel. Be sure to mix the samples well before loading, but
gently, as they will foam.
Using gel loading tips, load your samples in the following order:
Lane 1, 3, 4, 6, 7, 9 and 10: 1X SDS sample buffer
Lane 2: 20 µL Prestained protein marker
Lane 5: 60 µg wild type protein
Lane 8: 60 µg XntA1 protein
Run the gel at 150V (2 gels = 150V) for 1-1 ½ hours; until the dye runs off the bottom
edge of the gel.
Remove gel carefully from the gel apparatus. Use a razor blade to cut the stacking gel
portion away. Dispose of the stacking gel in the appropriate waste container.
Place the resolving gel into a plastic container and cover with Coomassie stain. Stain
while rocking gently overnight.
43
Next Day (Done for you):
9. Pour off the stain into original container.
10. Add destain to the gel. Rock the gel gently.
11. Discard the destain after ~1 hour and pour fresh destain over the gel. Continue to rock
at room temperature.
12. Again, remove the destain and replace with fresh destain. Rock gently overnight.
13. The Lab tech will save these gels in destain until the next lab.
3.5 Cutting up the gel and trypsinizing proteins in preparation for Mass Spec__
HAZARDOUS CHEMICAL INFO:
-Acetonitrile is poisonous. It also tends to leak from pipet tips when being measured. Always
wear eye protection and gloves when handling it or transferring it. Always dispose of
acetonitrile in its special waste receptacle. If you get acetonitrile on your gloves, change your
gloves.
The TA will demonstrate:
1. Clean a large glass plate with soap and water and rinse thoroughly with sterile water.
Rinse again with 95% ethanol. Let the plate dry.
2. Carefully remove the “chosen” gel from its container and place it on the glass plate.
3. Each group should label four 1.5 mL microcentrifuge tubes, two for control samples and
two for the mutant samples, with appropriate identification for each gel slice. For
example, C1 and C2 for control gel slices 1 and 2. There will be the same number of gel
slices for the control and mutant samples as there are students in the class (i.e. 12
students = 12 control gel slices and 12 mutant gel slices; 24 total).
4. Each gel slice will be cut using a razor blade that is cleaned with 95% ethanol before
using and between cutting each band.
5. Once the gel slice is cut out of the gel, the TA will slice each band into 1 mm x 1 mm
cubes and place these cubes in your labeled 1.5 mL microcentrifuge tube.
6. Add 900 µL of HPLC-grade water to each tube. Incubate at room temperature for five
minutes.
7. Centrifuge at high speed for 30 seconds. Using your pipettor set to 1000 µL, carefully
remove the water and discard it in the waste container provided. Use a new pipet tip
for each sample. Be careful not to lose the gel cubes in this process.
Note: The following steps use acetonitrile, which is poisonous. It also tends to leak from
pipet tips when being measured. Always wear eye protection and gloves when handling it or
transferring it. Always dispose of acetonitrile in its special waste receptacle. If you get
acetonitrile on your gloves, change your gloves.
8. Add 750 µL destain solution (50 mM ammonium bicarbonate, 50% acetonitrile) to each
tube. Close the tube cap and mix gently by inversion. Incubate the tubes at 37°C for 20
minutes.
9. At the end of 20 minutes inspect the gel. If it is still very blue, repeat the destain
process with more destain reagent. To do this, briefly centrifuge the tube, carefully
pipet off the liquid, add 750 µL new destain reagent and incubate for an additional 15
minutes. Make sure you put the discarded destain solution in the provided waste
receptacle. Repeat one more time if needed. Change gloves if you get acetonitrile on
them and wash skin with water if it contacts the acetonitrile.
44
10. When the gel sample is clear and no longer contains blue color, centrifuge the tube at
high speed for 30 seconds. Carefully remove all the destain solution and discard it in the
waste receptacle.
11. Add 100 µL of 100% acetonitrile to each tube. (The gel pieces should be entirely
immersed, if not add more.) The gel pieces will turn white as they dehydrate. Incubate
the tubes for 5 minutes at room temperature.
12. Centrifuge for 30 seconds and carefully pipet off all the acetonitrile. Discard in the
provided waste receptacle. Use a 200 µL pipettor to carefully remove the residual liquid
from the tube. If gel cubes are not completely opaque, repeat the process by adding
fresh acetonitrile and incubating for another 5 minutes. Also repeat the centrifugation
and removal step before moving forward.
13. Open lids and place tubes in Speed Vac for 5 minutes to dry samples completely. Close
the lids before proceeding to the next step.
14. Place the tubes on ice for 5 minutes. Add 30 µL ice cold Trypsin digestion buffer
solution to each tube. Incubate on ice for 5 minutes. Add more of the digestion buffer if
necessary to cover gel slices. Make sure the gel slices are completely immersed in the
solution.
15. Incubate the tubes on ice for an additional 30 minutes. Add more trypsin digestion
buffer if necessary.
16. Transfer the tubes to a 37°C incubator. Incubate the tubes overnight (16-18 hrs).
The Next Day (Please try to begin this process around noon):
17. Spin the tubes of gel slices for 1 minute at full speed. Transfer all of the peptide solution
(supernatant) to a properly labeled 0.5 mL tube.
18. Add 150 µL of 50% Acetonitrile / 2.5% formic acid to the tubes of gel slices and vortex.
Incubate for 45 min at RT.
19. Spin these tubes for 5 minutes at full speed and transfer the peptide solution
(supernatant) to their respective 0.5 mL sample tubes.
20. Add 100 µL of 100% acetonitrile to the tubes of gel slices and vortex. Incubate for 20
min at RT (Repeat if needed).
21. Spin for 5 minutes at full speed and transfer the peptide solution (supernatant) to the
respective 0.5 mL sample tubes. Save the tubes of gel pieces at room temperature for
later extraction if needed.
22. Dry the samples using the Speed Vac for 2 to 4 hours (the TA and technician will assist
with this process).
23. Your TA will bring your dried samples to the UVM Proteomics Core Facility for
processing.
3.6 Mass Spec
Tour the UVM Proteomics Core Facility in MLS 337 and meet to discuss how mass spec works.
3.7 Mass Spec Results Discussion_______________________________
Experienced proteomics personnel will discuss how to analyze the results you receive from the
Mass Spec.
45
3.8 Analyze results
Your TA and professor(s) will help you to analyze your results from mass spec, and determine
which proteins are differentially expressed.
46
Appendix A: Solutions Guide
GENERAL SOLUTIONS
Ampicillin (AMP) Stock
100 mg/ml stock solution: for example, 0.5 g ampicillin sodium salt into 5 mL dH2O.
Filter sterilize, and store at -20°C.
LB-AMP
100 µg/mL final concentration of AMP: a 1:1000 dilution of ampicillin stock into LB broth
(i.e. add 1 mL ampicillin stock (100 mg/mL) into 1 L LB Agar broth).
*NOTE: Ampicillin is heat-sensitive. LB agar broth must be cooled to 60°C after coming
out of the autoclave before the AMP is added. Setting a large water bath to 60°C and
letting the LB agar broth cool in the water after autoclaving, for an hour, is a good way
to ensure the LB agar doesn’t solidify and that it won’t be too hot for the AMP.
TE buffer – 1 L
Need (final conc.): 10 mM Tris-Cl, 1 mM EDTA
Make from liquid stocks of Tris-Cl and EDTA
5 mL 2 M Tris-Cl (pH 7.5)
2 mL 0.5 M EDTA (pH 8)
993 mL dH2O
2 M Tris-Cl (pH 8.0) – 1 L
177.6 g Tris-Cl
10.6 g Tris-base
In ~950mL sterile dH2O
**pH 8.0**
Bring up to 1 L with sterile dH2O
0.5 M EDTA (pH 8) – 100 mL
18.6 g EDTA disodium salt (FW= 372.2)
In ~75 mL sterile dH2O
Heat in microwave to dissolve salt
***bring pH to 8.0***
Bring up to 100 mL with sterile dH2O
50X TAE stock (pH 8.5) – 1 L
242 g Tris Base (FW= 121.14)
In ~700ml sterile dH2O
Carefully add 57.1 mL Glacial Acetic Acid
100 mL 0.5 M EDTA (pH 8.0)
Bring up to 1 L with sterile dH2O
pH 8.5, but no adjustment needed
*Dilute 50X TAE stock 1:10 for a 5X stock*
47
6X DNA loading buffer – 100 mL
0.25 g Bromophenol Blue
40 g Sucrose
100 mL dH2O
λ Hind III marker
Want a final concentration of 100 ng/µL
Make: 96 µL λ Hind III marker (Invitrogen stock)
320 µL TE
68 µL 6X DNA loading buffer
20X SSC (pH 7) – 1 L
175.3 g NaCl
88.25 g Na3 Citrate•2H2O
1 L dH2O
**pH 7.0**
2X SSC – 100 mL
10 mL 20X SSC
90 mL dH2O
10X TBS (pH 7.4) – 1 L
25.1 g Tris HCl
4.8 g Tris Base
80 g NaCl
800 mL of H2O.
Bring up to 1 L with high purity distilled or deionized water.
Once prepared, TBS is stable at 4°C for 3 months.
10X TBS, 1% Tween-20
10 mL Tween-20 (use large orifice tips or slow serological pipetting to pick up Tween)
1 L 10X TBS
Phosphate Buffer Saline (PBS) – 500 mL
4.0 g NaCl
0.1 g KCl
0.72 g Na2HPO4
0.12 g KH2PO4
In ~400ml sterile dH2O
**pH 7.4**
Bring to 500 mL with sterile dH2O
48
MODULE 1
1.1: Isolation of plasmid DNA
Solution I
0.50 g D-Glucose
0.625 mL 2M Tris-Cl (pH 8)
1 mL 0.5M EDTA
Add dH2O to make total volume 50 mL
**add 5 mg/ml lysozyme just before use**
Solution II
2 mL 1M NaOH
1 mL 10% SDS
7 mL dH2O
**Prep fresh**
3M Potassium Acetate (100 mL)
29.4 g potassium acetate salt (F.W. = 98.14 g/M)
29.5 mL glacial Acetic Acid
Bring volume close to 100 mL using dH2O
Add KOH pellets until pH=4.8
Bring to volume and store at 4°C.
Heat-treated RNase A (100mg/ml)
Dissolve 100 mg (0.1 g) of pancreatic RNase A in 1 mL 10 mM Tris-Cl/15 mM NaCl. Store
at -20 °C. Before use, heat RNase A in 100°C heat block for 15 minutes and allow tubes
to cool slowly to RT.
10mM Tris-Cl/15mM NaCl
0.1576 g Tris-Cl
0.0876 g NaCl
100 mL dH2O
Salt Saturated (SS) Phenol
Tris buffered Phenol pH 6.6/7.9
8-Hydroxyquionoline added until dark yellow/orange color
Chloroform:Isoamyl Alcohol (24:1)
480 mL Chloroform
20 mL Isoamyl Alcohol
TE buffer
See general solutions section
49
1.4: Transformation
50 mM CaCl2
0.73 g CaCl2
100 mL dH2O
** Make fresh**
Transformation buffer
1 mL 100 mM CaCl2
1 mL 100 mM Tris (pH 8.0)
1 mL 100 mM NaCl
7 mL dH2O
**Store at 4°C**
1.6: Secondary selection of transformed bacteria
0.145 M Sterile Saline (pH ~7)
4.25 g NaCl
500 mL dH2O
** Autoclave to sterilize**
1.8: Cracking gel
Cracking Buffer (pH 6.8)
0.788 g Tris-Cl
1.0 g SDS
0.058 g Na2EDTA•2H2O
13.6 g Sucrose
0.1 g Bromophenol Blue
100 mL dH2O
** pH 6.8**
1.9: Biotin labeling of DNA
3 M Sodium Acetate (pH 4.8)
24.6 g Sodium Acetate
100 mL dH2O
** pH 4.8**
50
1.11: Southern blot
Cracking Buffer
See 1.8 above
0.5 M NaOH/0.8 M NaCl
20 g NaOH
46.75 g NaCl
1 L dH2O
0.5 M Tris/1.5 M NaCl (pH 7)
250 mL 2 M Tris-base solution (pH 8)
87.6 g NaCl
750 mL dH2O
** pH 7**
10X SSC
See general solutions for 20X SSC
1.13: Hybridization of Southern Blot
Prehybridization Solution (per group, prep fresh)
5 mL Formamide
2.5 mL 20X SSC
0.5 mL 100X Denhardt’s solution (doesn’t keep more than 24 hours!)
0.25 mL 1 M Phosphate Buffer
200 µL Herring sperm DNA (2mg/mL, made fresh), freshly denatured
Hybridization Solution (per group, prep fresh)
4.5 mL Formamide
2.5 mL 20X SSC
0.1 mL 100X Denhardt’s solution (doesn’t keep more than 24 hours!)
0.4 mL 1 M Phosphate Buffer
1.5 mL dH2O
200 µL Herring sperm DNA (2mg/mL), freshly denatured
Biotin-labeled probe DNA
100X Denhardt’s Solution
0.2 g Ficoll
0.2 g Polyvinylpyrrolidone
0.2 g Bovine Serum Albumin (BSA)
10 mL sterile dH2O
**Doesn’t keep more than 24 hours**
51
1.14: Detection of DNA
2X SSC/0.1% (w/v) SDS
50 mL 20X SSC
450 mL dH2O
0.5 g SDS
0.2X SSC/0.1% (w/v) SDS
5 mL 20X SSC
495 mL dH2O
0.5 g SDS
0.16X SSC/0.1% (w/v) SDS
4 mL 20X SSC
496 mL dH2O
0.5 g SDS
1.15: Development of Blot
Buffer 1: Final Concentration: 0.1 M Tris-Cl, 0.15 M NaCl
8.7 g NaCl
15.76 g Tris-Cl
Bring to 1 L with dH2O
Buffer 2: 3% (w/v) BSA in Buffer 1
3 g BSA per 100 mL Buffer 1
**Doesn’t keep more than 24 hours, prep fresh**
SA-AP: **Needs to be made immediately before use**
1 µL SA-AP per 1 mL Buffer 2 (approx. 10 mL needed per group)
Buffer 3
Final concentration: 0.1 M Tris-Cl pH=9.5, 0.1 M NaCl, 50 mM MgCl2
15.764 g Tris-Cl
5.844 g NaCl
10.15 g MgCl2
Bring to 1 L with dH2O
1.17: PCR
20 µM Primer Dilutions (from 500 µM stock primers)
5 µL 500 µM stock
120µL dH2O
**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in smaller
batches**
52
10 mM dNTP mix (from 100 mM individual dNTP stocks)
500 µL dCTP
500 µL dTTP
500 µL dATP
500 µL dGTP
3 mL dH2O
*1 µL of 10 mM dNTP mix per 50µL reaction*
MODULE 2
2.2 PCR
20 µM Primer Dilutions (from 500 µM stock primers)
5 µL 500 µM stock
120 µL dH2O
**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in smaller
batches**
10 mM dNTP mix (from 100 mM individual dNTP stocks)
500 µL dCTP
500 µL dTTP
500 µL dATP
500 µL dGTP
3 mL dH2O
*1 µL of 10 mM dNTP mix per 50 µL reaction*
2.7 Induce Bacteria and Feed to Paramecium
0.2 M IPTG
750 µL dH2O
37.5 mg IPTG
Wheat culture
Wheat grass tea buffered with:
3.75 mM Na2HPO4*7H2O
3 mg/L Stigmasterol
Dryl’s solution – 1 L (final concentrations)
1 mM Na2HPO4
1 mM NaH2PO4
1.5 mM CaCl2
2 mM Sodium Citrate
pH 6.8
Stigmasterol
5 mg/mL dissolved in 100% ethanol
53
2.9 Harvest cells and Isolate RNA
Resting Solution (final concentrations)
1 mM Calcium citrate
~1.3 mM Tris base
5 mM KCl
Adjust pH to 7.03 using Tris base
2 mM BaCl2 (final concentrations)
1 mM calcium citrate
~1.3 mM Tris base
2 mM BaCl2
Adjust pH to 7.03 using Tris base
2.10 Making cDNA
50 µM Oligo dT20
Dilute 1:10 from 500 µM primer stock
Primer Sequence: 5’ CGGCTCGAGTTTTTTTTTTTTTTTTTTTT 3’
10 mM dNTP mix
(See 2.2 above)
2.11 Template Control and Endogenous RT-PCR
20 µM Primer Dilutions (from 500 µM stock primers)
5 µL 500 µM stock
120 µL dH2O
**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in smaller
batches**
MODULE 3
3.0 Preparation of Cilia
Dryl’s solution – 1 L (final concentrations)
1 mM Na2HPO4
1 mM Na2H2PO4
2 mM Sodium Citrate
1.5mM CaCl2
pH 6.8
54
STEN Buffer – 100 mL
17.12 g Sucrose
0.316 g TrisCl
0.068 g Na2EDTA
0.036 g NaCl
100 mL dH2O
*pH to 7.5 and fill
**store at 4C
Cilia Shock Buffer (final concentrations)
180 mM KCl
60 mM CaCl2
Membrane Buffer – 50 mL
0.331 g Tris-Cl
0.048 g Tris base
0.186 g KCl
0.051 g MgCl2•6H2O
0.019 g EGTA
50 mL dH2O
*pH to 7.4*
**add 1% Triton X-100 before use
Leupeptin
Dissolve in dH2O 1 mg/mL
Store at -20C in 1 mL aliquots
*Stable for 6 months*
Pepstatin A
Dissolve in methanol 1 mg/mL
(5 mL methanol into 5 mg bottle and shake)
Store at -20C in 1 mL aliquots
*Stable for 1 month*
100 mM PMSF stock (Phenylmethylsulfonyl fluoride)
0.871 g PMSF
50 mL 100% ethanol
Add ~1.75 g of molecular sieves to each bottle to absorb any water
*inactive in 30 minutes @ 4C in aqueous solution*
Preparation of Membrane Buffer with Protease Inhibitors
1 mL Membrane Buffer
2 µL Pepstatin
2 µL Leupeptin
10 µL PMSF
2 µL IAA (0.02 g/mL)
*Add these immediately before use*
55
Tris EDTA – 100 mL
0.0121 g Tris Base
0.0034 g Na2EDTA
100 mL dH2O
*pH 8.3*
3.1 Ultracentrifugation
10 mM Tris pH 8.0
5 mL 1M Tris, pH 8.0
495 mL dH2O
66% Sucrose
171 g Sucrose
90 mL 10 mM Tris pH 8.0
Make the night before and place in shaker at 37o C overnight to dissolve
3.2 Preparation of Resolving Gel
4X Resolving Buffer (pH 8.9) – 100 mL
18.17 g Tris base
10 g SDS (measure in hood)
100 mL dH2O
Adjust pH to 8.9 using conc. HCl
3.4 SDS-PAGE
4X Stacking Buffer (pH 6.8) – 100 mL
6.055 g Tris base
0.4 g SDS
100 mL dH2O
Adjust pH to 6.8 using conc. HCl
10X SDS Sample Buffer – 25 mL
15.6 mL 8X TrisCl/SDS (pH 6.8)
10 mL Glycerol
2.0 g SDS
5 mg Bromophenol blue
*Add 1 µL of β-Mercaptoethanol to 10 µL of 10X SDS Sample Buffer*
56
8X TrisCl/SDS (pH 6.8)
6.05 g Tris base dissolved in 35 mL dH2O
Adjust the pH to 6.8 with concentrated HCl, and bring to 50 mL volume.
0.4 g SDS
5X Running Buffer
7.55 g Tris Base
36 g Glycine
*Bring solution to 500 mL volume and stir. pH should be 8.3 but don’t adjust.*
Add 2.5 g SDS.
**Store at 4°C** Dilute to 1X before using
Coomassie Blue Stain – 500 mL
200 mL Methanol
50 mL Glacial Acetic Acid
1 g Coomassie Blue
250 mL dH2O
Coomassie Blue Destain – 1 L
200 mL Methanol
75 mL Glacial acetic Acid
725 mL dH2O
3.5 Band Cutting and Trypsinization of Gel Pieces
100 mM ammonium bicarbonate stock – 250 mL
1.977 g ammonium bicarbonate
Bring to volume with dH2O
Destain solution
50 mM ammonium bicarbonate
50% acetonitrile
Trypsin Digestion buffer – enough for ~26 samples
42 µL Trypsin (20 µg/50 µL)
560 µL 100 mM ammonium bicarbonate stock
70 µL 100% acetonitrile
700 µL dH20
Keep on ice!
57
Appendix B: Sterile Technique
It is very important in microbiology and genetics to work with pure cultures. Unfortunately, this
is difficult. The world around us is covered with microorganisms. Microorganisms are even
carried on dust particles in the air. In order to protect sterile broth, plates, solutions and pure
cultures from the microbes all around us, we must practice sterile (aseptic) technique. This
simple means that sterile surfaces or sterile media must be protected from contamination by
microbes in the air or residing on non-sterile surfaces. A simple example of the problem is that
a sterile petri plate can become contaminated with bacteria when the lid is removed. In sterile
technique, only sterile surfaces touch other sterile surfaces and exposure to the air is kept to a
minimum.
In the classroom, you often need to practice sterile technique when you inoculate a pure
culture of a microorganism into fresh medium. Sometimes this is a transfer to a tube of liquid
broth and at other times, it is a transfer to a petri plate-containing agar. While there are other
circumstances that require sterile technique, these are the most common and they will be
described in more detail on the pages that follow.
Here are a few simple techniques to help you along your way:
 Always close tip boxes and tube containers.
 Always place caps face-down on counters and replace them promptly onto bottles and
secure them.
 Never leave plates open to the air; only remove the lids to spread something or remove
something.
 Do not hold plates close to your face.
Appendix C: Spread Plate Technique
1. Dispense the appropriate volume of sample into the center of a sterile agar plate.
2. Dip the glass spreader (aka “hockey stick”) in alcohol or gently spray alcohol on the
hockey stick over the sink.
3. Pass the spreader through the flame of a Bunsen burner to burn off the alcohol. (This
sterilizes the spreader.)
***IMPORTANT***
Keep your hand above the spreader at all times (i.e., keep it tilted toward the floor) or
flaming alcohol may roll toward your hand.
4. Cool the spreader by touching it to the agar where there is no sample.
5. Spread your sample over the entire surface of the agar.
6. Sterilize the spreader before putting it back on the bench.
58
Appendix D: Use of a Rainin Pipettor
Take note:
 Never rotate the volume adjustor beyond the upper or lower range of the pipettor, as
stated by the manufacturer.
 Never use the pipettor without the tip in place; this could ruin the precision piston that
measures the volume of fluid.
 Never lay down the pipettor with filled tip; fluid could run back into the piston.
 Never let plunger snap back after withdrawing or ejecting fluid; this could damage the
piston.
 Never immerse the barrel of the pipettor in fluid.
 Never flame your pipettor or pipette tips.
If you drop your pipettor, the precision piston system can be damaged; therefore, if your
pipettor is dropped, be sure to check the pipetting accuracy has not been affected.
Recommended Volume Ranges:
Model p10: 0.5-10 μL, the number after the decimal point is in red
Model p20: 2-20 μL, the number after the decimal point is in red
Model p200: 20-200 μL, there is no decimal point
Model p1000: 200-1000 μL, the numbers after the decimal point are in black
Pipetting Directions – Method
1. Set the desired volume by holding the pipettor body in one hand and turning the
volume adjuster knob until the correct volume shows on the digital indicator. Approach
the desired volume by dialing downward from a larger setting.
2. Press tip onto shaft by a slight twisting motion.
3. Depress the plunger to FIRST POSITIVE STOP. This part of the stroke is the calibrated
volume displayed on the digital micrometer.
4. Holding the pipette man vertically (never more than 20˚ from vertical), immerse the tip
just below the level of the liquid.
5. Allow the pushbutton to return SLOWLY to the up position. Move the tip so that it stays
slightly below the level of the liquid as you draw up.
6. Wait one to two seconds to ensure that the full volume of sample is drawn up into the
tip.
7. Withdraw the tip from the sample liquid.
8. To dispense the sample, place the tip end against the sidewall of the receiving vessel
and depress the plunger to the FIRST STOP. Wait one to two seconds. Then depress the
plunger to the SECOND STOP, expelling any residual liquid in the tip.
9. With the plunger fully depressed, withdraw the pipettor from the vessel carefully with
the tip sliding along the wall of the vessel.
10. Let the plunger return slowly to the UP position. If an air bubble is observed, re-pipette
the sample.
11. Pre-rinsing the tip with the liquid being pipetted is recommended. A significant film
may be retained on the inside wall of the tip, resulting in an error. Since the film
59
remains relatively constant in successive pipettings with the same tip, refilling the tip a
second time and using this quantity as the sample may obtain good reproducible results.
12. Discard the tip by depressing the tip ejector button smartly in the appropriate waste
container.
60
APPENDIX E:
Label
S1
S2
S3
S4
S5
S6
S7
S8
PIERCE PROTEIN ASSAY for Module 1 Pipetting Exercise
OD Value
Series 1
Series 2
L Alb (Stock L dH2O mL Dye [Alb g/mL]
2 mg/mL)
0
100
2
0
2.5
97.5
2
50
5.0
95.0
2
100
7.5
92.5
2
150
10.0
90.0
2
200
12.5
87.5
2
250
15.0
85.0
2
300
25.0
75.0
2
500
Label
L
Sample
L
dH2O
mL
Dye
Unknown 1
Unknown 2
100
100
0
0
2
2
Unknown 1
Unknown 2
100
100
0
0
2
2
OD
Value
g/mL in
Cuvette
g/mL
Original
Solution
61
APPENDIX F: GST Plasmid Map
Websites: www.GEHEALTHSCIENCES.com
https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/131471676253
6/litdoc28956795_20140411174609.pdf
62
APPENDIX G: L4440 Plasmid Map
63
64
APPENDIX H: Streak Plate Method
65
APPENDIX I: Frequently Used DNA/Protein Markers
Lambda DNA-Hind III Digest
100 bp DNA Ladder
Prestained Protein Marker
1Kb DNA Ladder
66
APPENDIX J: Southern Blot Assembly
Paper Towels
10 CM
Whatman Paper
Nitrocellulose
Agarose Gel
Whatman Paper
Wick
10X SSC
Platform
67
APPENDIX K: PCR Reagents and Conditions for 1.17
Cycling Program: GST
95°C
5 min
Initial
Denaturation
95°C
50°C
72°C
1 min
1 min
1 min
5X
95°C
51°C
72°C
1 min
1 min
1 min
25X
72°C
4°C
10 min
HOLD
Final elongation
***************
SAMPLES
Initial Stock
Concentration
25 mM
10 mM
20 µM
20 µM
5 U/µL
Components
1
2
3
5
3
colonies
5 µL
6
7
1 µL
5 µL
4
3
colonies
5 µL
Template DNA*
10X Buffer
1 µL
5 µL
1 µL
5 µL
1 µL
5 µL
1 µL
5 µL
MgCl2
dNTPs
Forward Primer
Reverse Primer
Taq Polymerase
0.5 µL
1 µL
1 µL
1 µL
0.5 µL
1 µL
1 µL
1 µL
1 µL
0.5 µL
3 µL
1 µL
1 µL
1 µL
0.5 µL
3 µL
1 µL
1 µL
1 µL
0.5 µL
3 µL
1 µL
1 µL
1 µL
0.5 µL
3 µL
1 µL
XXX
1 µL
0.5 µL
3 µL
1 µL
1 µL
XXX
0.5 µL
dH2O
*NOTE: Samples 1-3, 6 & 7: Use PLASMID DNA
Sample 4: Transformed Colonies
Sample 5: Non-Transformed Colonies
DESIRED FINAL VOLUME: 25 µL
68
APPENDIX L: Sucrose Gradient Calculations
How to make __% Sucrose from 66% Sucrose
Add __ mL of
66% sucrose
Add __ mL 10 mM
Tris, pH 8.0
55% Sucrose
8.00
2.00
45% Sucrose
6.35
3.65
20% Sucrose
2.45
7.55
How to Create the Sucrose Gradient:
First add 0.7 mL 66% Sucrose,
Then add 1.4 mL 55% Sucrose,
Then add 1.4 mL 45% Sucrose,
Finally add 0.7 mL 20% Sucrose.
0.7 mL 20% Sucrose
1.4 mL 45% Sucrose
1.4 mL 55% Sucrose
0.7 mL 66% Sucrose
69
APPENDIX M: Sucrose Gradient Tubes after Ultracentrifugation
20%
Ciliary membrane
45%
Partial axoneme
55%
Pure axoneme
66%
70
APPENDIX N: Protein Gel Plate Setup
Mini-PROTEAN®
71
APPENDIX O: PIERCE PROTEIN ASSAY for Module 3
Label
S1
S2
S3
S4
S5
S6
S7
Label
L Alb (Stock
2 mg/mL)
0
2.5
5.0
7.5
10.0
12.5
15.0
L
Sample
L dH2O
100
97.5
95.0
92.5
90.0
87.5
85.0
mL
Dye
2
2
2
2
2
2
2
L
dH2O
mL
Dye
[Alb
g/mL]
0
50
100
150
200
250
300
OD Values
OD Value
Series
Series
A
B
Average
OD Value
Average
g/mL in
Cuvette
g/mL
Original
Solution
2
2
2
2
Label
Average g/mL
Original Solution of
each treatment
g /L
Original solution
L of protein that
equates to 60 g
total
WT
XntA1
72
APPENDIX P: Protein Gel Running Setup
Mini-PROTEAN®
73
APPENDIX Q: Graphing your protein data in Excel
1.
2.
3.
4.
5.
6.
Enter your standard concentrations into column A.
Enter the measured ABS into columns B and C.
Calculate average of each reading into column D.
Highlight columns A and D.
On the insert tab, choose scatter plot with no lines.
Right click on the graph data and choose trend line.
a. Click on linear
b. Choose Display equation on chart
c. Choose Display R-squared value on chart
7. Label axes, and title.
Adding Error Bars
8. Enter standard deviation formula into column E
a. Place the curser into E1 and clicking on Formulas,
b. Select insert function,
c. Select STDEV,
d. Click OK,
e. Click the square in the number 1 line.
f. Highlight B1 and C2 cells.
g. Click the square in the Number 1 line.
h. Click OK.
i. Copy formula down column E.
9. Click onto the chart.
10. In the chart tools at the top of the sheet, choose Layout
11. Under layout, choose Error Bars,” more error bars options”.
12. Choose both + and – error bars.
13. Choose Custom and specify value.
14. Highlight the standard deviations in column E for both plus and minus.
15. Click OK.
16. Click close.
74
APPENDIX R: RPM to G-Force Conversions
Equipment
Damon IEC clinical HN S-II centrifuge
Damon IEC clinical HN S-II centrifuge
Beckman J2-21 w/JA 14 rotor (250 mL tubes)
Beckman J2-21 w/JA 17 rotor
Beckman J2-21 w/JA 17 rotor
Beckman J2-21 w/JA 17 rotor
Beckman J2-21 w/JA-17 rotor
Beckman J2-21 w/JA-20 rotor
Beckman J2-21 w/JA-20 rotor
IEC Centra 7 Desktop w/ 15 mL tubes
IEC Centra 7 Desktop w/ 50 mL tubes
Eppendorf Centrifuge 5702
Eppendorf Centrifuge 5702
Eppendorf Centrifuge 5415 C
Sorvall Legend Centrifuge
Ultracentrifuge
Ultracentrifuge
RPM
3/4
Full
5,000
5,000
9,500
15,000
17,000
14,500
19,500
2,800
2,800
2,800
2,800
14,000
14,000
45,000
21,600
G-Force (RCF or × g)
500
850
3,840
3,440
12,400
31,000
39,000
25,400
46,000
1,098
1,098
1,120
1,180
15,980
18,800
208,000
45,000
75
76
Notes
77
Notes
Notes
78
Notes
Notes
79
Notes
Notes
80
Notes
81