BIOLOGY 204/205 Advanced Genetics Laboratory
TABLE OF CONTENTS
Introduction……………………………………………………………………..………….…p.2
MODULE 1: Recombinant DNA…………………………………………...…….…..p.9
MODULE 2: RNA Interference………………………………………………………..p.22
MODULE 3: Proteomics………………………………………………………………….p.34
Appendix A: Solutions Guide……………..…………………………………..……...p.42
Appendix B: Sterile Technique………………………………………………….......p.54
Appendix C: Spread Plate Technique…………………………………………...…p.55
Appendix D: Pipette Use……………………………………………………………......p.55
Appendix E: Pipette Exercises …..…………………...….…………………….......p.57
Appendix F: GST Plasmid Map………………………………………………………..p.58
Appendix G: Plasmid Map …………...………………………..……………….....…p.59
Appendix H: DNA/Protein Markers………………………..………………......…p.60
Appendix I: Streak Plate Method…………………………………………………...p.61
Appendix J: PCR Reagents and Conditions for 1.17……………..……..….p.62
Appendix K: Sucrose Gradient Calculations …………………………….…....p.63
Appendix L: Sucrose Gradient Tubes after Ultracentrifugation ……..p.64
Appendix M: Protein Gel Plate Setup ….…………………………………………p.65
Appendix N: Pierce Protein Assay for Module 3 ………………………......p.66
Appendix O: Protein Gel Running Setup …………………………….……….…p.67
Appendix P: RPM to G-force Conversions………………………….…….……..p.68
1
BIOLOGY 204/5
Advanced Genetics Laboratory I and II
--- Introduction --Module 1 Recombinant DNA/Bacterial Transformation
This module gives you some of the experience you would receive if you were to subclone 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. To purify a plasmid and transform E. coli with the plasmid.
2. To demonstrate that the transformants carry the plasmid by characterizing the
transformants’ phenotypes.
3. Analyzing the size of the DNA plasmid in a cracking gel.
4. Hybridization 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
In this module, a specific target gene product of Paramecium tetraurelia is depleted
using an RNAi feeding method. You will isolate RNA from the paramecia and determine
if there has been down regulation of the gene product in the RNAi treated population
compared to the control.
Goals:
1.
2.
3.
4.
5.
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
Determine the level of target endogenous mRNA by semi-quantitative Reverse
Transcriptase- Polymerase Chain Reaction (RT-PCR)
2
Module 3 Proteomics
In this module you will compare the proteins found in wild type and mutant paramecia
cilia. You will run a one dimensional polyacrylamide gel, cut out bands of interest, and
compare the proteins present in those bands using mass spec.
Goals:
1. Use ultracentrifugation to isolate ciliary membranes from wild type and mutant
cells
2. Conduct a Pierce Protein Assay to determine the concentration of cilia 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
3
Biology 204/205 Advanced Genetics Laboratory
Grading Policy
Biology 204 and Bio 205 are four credit courses. You will complete Modules 1 & 2 and a
grant writing exercise during the fall semester for Bio 204. For Bio 205 in the spring
semester, you will complete two modules and give a group presentation on the results.
The emphasis of the course 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 return to lab outside of the scheduled class time, usually at their own
convenience, to perform a short manipulation. 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!
Grading
Your grade will be based on the following components, each with approximately equal
weight:
1. Performance in laboratory
2. Discussion of experiments in class
3. Notebook (these will be checked weekly)
4. Laboratory report
5. Lab meeting presentations
6. Oral Exam
7. 10% will be deducted from grade per day for late assignments.
8. Presentations must be in by 4pm on the assigned date. 10% will be deducted
from grade for late Presentations.
Note: It is important to read over the procedures in the laboratory manual before
coming to class. Be prepared to start work after an introduction by the instructor or TA.
Check the laboratory calendar so that you know when each module will occur.
4
Performance in Laboratory Guidelines
1. Attendance
a. Includes arriving on time and staying until work is complete
b. Coming in willingly on “off” days when necessary
2. Working well and cooperatively with lab partner(s)
a. Simulates behavior in collaborative research groups behavior
b. Is behavior disruptive or helpful, distracting, professional?
3. Reading the lab manual in advance and arriving prepared for the day’s methods
a. Working efficiently
b. Following through
c. Being engaged, even when not physically performing experiments
4. "Lab Citizenship"
a. Such as following the safety rules, cleaning up, labeling properly, putting
materials away, etc
5. Attitude and willingness to participate in experiments
Laboratory Notebook Guidelines
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Hard cover, bound notebook; no loose-leaf
Record in blue or black ink
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
Neat, orderly, complete
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: What was done and why, who suggested it,
who did it and when it was done, what results were obtained and what conclusions
were drawn.
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Laboratory Report Guidelines
You will be asked to write a formal report of one of the results from one of the lab
modules. You will prepare this report as you would prepare a manuscript for
publication, with 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 in its entirety should not exceed 10 pages in length. It should be printed
double-spaced, with no less than 1-inch margins. It must be in 12-point size in a
common font. Each section except the introduction should be started by 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.
Abstract: 250 words summarizing the experiment.
Introduction: 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 exercise should be included in the introduction. Recommended
length: 1.5 pages.
Methods: The methods section must be detailed enough to allow the reader to repeat
the exercise. You do not need to repeat the detailed description of the protocols in the
laboratory manual, but you should refer to the methods in the manual (Format:
Laboratory manual Page x-y) at the appropriate points. Recommended length: 3 pages.
Results: The results section reports upon what happened during the exercise. You must
include photocopies of the final gels and provide in tabular form other measurements
and data you collected. Each figure should have a brief descriptive caption, 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 pages.
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Discussion: 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.
Safety in the Laboratory
General Rules:
1. Disinfect your bench top with a 10% bleach solution when you arrive and when
you finish lab.
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.
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. Try to avoid wearing baggy, loose clothing that can interfere with your
experiment and may catch on fire.
12. Extinguish burners as soon as you finish using them.
13. All Chemical Safety and MSDS information is located in the binder on the back of
the door.
14. If you are unsure about a procedure, just ask.
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Pipetting
1. Acquaint yourself with the various denominations of pipettors in an attempt to
avoid mistakes, particularly when working under time pressure.
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/99 0µL
8
Module 1
Recombinant DNA
Please refer to page 2 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.
1.0 Overnight (ON) Bacterial Culture (Done for you)
1. The lab tech will add 2.5 ml of cells previously grown ON to 125 ml LB amp
medium (per group).
2. The cells will grow with shaking at 37C ON.
1.1 Isolation of Plasmid DNA
HAZARDOUS CHEMICAL INFO:
-Salt-Saturated 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) 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 125ml 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 5ml of Solution I containing 5mg/ml lysozyme.
4. Transfer to a 30ml polycarbonate screw top Oakridge centrifuge tube. Let stand
at room temperature for 5 minutes.
5. Add 10ml of freshly made Solution II. Place the cap on the tube and mix the
contents by inverting the tube several times. Mix gently. Let stand on ice for 10
minutes.
6. Add 8ml of ice-cold 5M 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.
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 10ml pipet and transfer all to a
30ml glass screw top tube. Only take clear supernatant.
10. Add at least 500µL of heat treated RNase A to each tube. Please use all of the
RNase A provided.
11. Incubate at 37ºC for 30 minutes in Innova 4000.
12. In the chemical flow hood, add one volume of SS (salt saturated) phenol using
glass pipettes. (Note the yellow color which helps you identify the phenol phase
9
in the next step.) Your tubes can be no more than 2/3 full including the addition
of the phenol, so divide your original solution as necessary into 3 or 4 conical
tubes, using glass pipettes.
SAFETY NOTE: Phenol can cause severe burns to skin and damage clothing.
Gloves, safety glasses, and a lab coat should be worn when working with
phenol. All manipulations should be carried out in a 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 is 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 30ml 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 30ml glass tube. The tube cannot be more than 2/3 full, so
you may have to use more than one 30ml glass tube. Make your calculations
before adding the ethanol!
16. Mix and allow it to precipitate in the -80o freezer overnight.
Next Day
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 1.5ml microfuge tube.
21. Microcentrifuge for 5 minutes at 14,000 rpm. Pipet out the ethanol; add 1ml
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 0.3ml 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:
10
-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.
1.
2.
3.
4.
Prepare 300ml 1X TAE from 5X TAE stock.
Dissolve 0.35g agarose in 50ml 1X TAE buffer to make a 0.7% gel.
Microwave on high for 1 minute.
Swirl the flask and make sure all of the agarose is dissolved. If not, microwave
until it is. Remove flask with a hot mitt.
5. Place the running tray into the gel-casting tray. Add comb.
6. 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).
7. While your gel is setting, thaw out one tube of your plasmid DNA on ice.
Just before you are ready to load the gel, heat the λ Hind III marker for 7
minutes in the 65°C hot block. – PLACE ON ICE IMMEDIATELY.
8. Mix 4µL of 6X DNA sample buffer with 20µL plasmid DNA on a piece of Parafilm.
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. Be prepared to load the gel quickly—you do not want your DNA to diffuse into
the running buffer.
11. Load 24µL of plasmid DNA sample and 20µL of λ Hind III marker in the wells; put
the lid on the box so that the DNA will run toward the red electrode.
12. Run the gel at 100V for ~1 hour.
13. Stain the gel for approximately 15 minutes in ethidium bromide, and destain in
water for 5 minutes.
14. 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.
15. Leave the gel in destain to be discarded later.
16. 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)
1.4 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 transform.
11
1. Divide broth culture into 2 sterile 30ml 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 resuspend gently the pellet in 3ml ice-cold 50 mM
CaCl2; place on ice for 5 minutes.
7. Dispense 2 aliquots of 0.3 ml cells in ice-cold 1.5ml 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. The second tube will not contain 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. 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 (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 in
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 6 well isolated colonies from the transformed plates. Streak each colony
on half of an LB amp plate.
3. Choose 2 well isolated colonies from the control (non-transformed) plate
provided. Streak each colony on one half of an LB plate.
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4. Incubate the plates overnight at 37°C.
5. Wrap the old plates in Parafilm and refrigerate.
1.6_Secondary selection of transformed bacteria_________________
1. Transfer 4 well-isolated colonies from 4 different transformed streaks and 2
control colonies into separate 1ml aliquots of sterile saline. Refrigerate the old
plates.
2. For the transformed bacteria, streak 1 loopful of saline/bacteria suspension onto
½ of an LB amp plate. Do this for each of the 4 samples.
3. For the control cells, streak 1 loopful of the saline/bacteria suspension onto ½ of
an LB plate. Be sure to label plates clearly!
4. Incubate at 37°C overnight; remove and refrigerate the next day.
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. Using sterile tweezers, select a sterile toothpick.
3. Choose 2 LB amp plates from Day 1.6 that show the best growth. With the
toothpick, select one colony from the Day 1.6 plate and “fill in” the square on
the agar on the plate. Repeat for the 2nd colony using a new toothpick.
4. Repeat the procedure for the control, but use a fresh LB plate.
5. Incubate at 37°C overnight for at least 24 hrs, but less than 36 hrs.
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. Make 300ml 1X TAE.
2. Prepare 0.7% agarose gel.
3. Use a sterile toothpick to scrape bacteria from the plates prepared the day
before. Add bacteria from each square to 250µL of cracking buffer (Two squares
for one tube of 250µL of cracking buffer). Do this for transformed and nontransformed cells (you should have a total of 2 microcentrifuge tubes). Vortex
tubes to mix well.
4. Incubate at 37°C in the hot water bath for 25 minutes.
5. Centrifuge for 15 minutes at 14,000 rpm.
6. 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).
7. Load the gel slowly and carefully:
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Lane 1: 20µL Hind III marker (Heat in 65C hot block for 7 minutes before
loading)
Lane 2: 20µL plasmid DNA solution (10µL plasmid DNA + 4µL 6X DNA sample
buffer + 6µL 1X TAE)
Lane 3: Transformed supernatant
Lane 4: 50µL Cracking buffer only
Lane 5: Non-transformed supernatant
Note: Load as much transformed and nontransformed supernatant as possible
(A well-formed well can hold ~50 L).
8. Run the gel for 1 hour at 100 volts.
9. Stain with ethidium bromide, destain, and photograph. 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 0.5ml tube (on ice):
dH2O
128µL
dNTP mix
28µL
1X DNase I Buffer
19.9µL
DNase I
0.1µL
Plasmid DNA
4µL
DNA Polymerase I
20µL
3. Mix well and centrifuge for 5 seconds at desktop spinner.
4. Allocate 50µL into 4 tubes.
5. Incubate at 15°C for 2 hours in thermocycler.
6. Add 5µL Stop Buffer to each tube and mix.
7. Incubate tubes at 65°C for 5 minutes in thermocycler.
Part B: Purification of DNA probes
1. Transfer liquid to consolidate solution from 4 tubes into one tube.
2. Add 4µL 10% SDS to tube and mix.
3. Add 110µL Chloroform and 110µL SS Phenol to an empty 1.5ml
microcentrifuge tube.
4. Transfer DNA solution to chloroform phenol tube. Vortex 2 minutes and
then centrifuge for 2 minutes at 14,000 rpm
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5. Collect the top layer of liquid and transfer to a fresh 1.5ml tube. Discard
remaining liquid into waste container.
6. Add 220µL 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 40µL 3M Sodium Acetate (pH 4.8) and 800µL 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.
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 1ml 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, resuspend probe in 12µL 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 toothpick, 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 use a fresh toothpick.
3. Repeat steps one and two, this time using an LB plate and the Day 1.6 nontransformed cells.
4. Incubate both plates for at least 24 hours.
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 (same as Day 1.8, except add 20ul of plasmid mixed with 4ul 6X
DNA sample buffer to the plasmid lane). Do not forget control lane!
2. Stain with ethidium bromide, briefly destain, and examine the gel.
15
3. Make sure to destain the gel for approximately 5 minutes before denaturing.
4. Photograph the gel before destaining completely—you will use this photograph
later to compare to the results of your southern blot.
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. While the gel is rinsing, cut and hydrate the nitrocellulose filter for 3 minutes in
dH2O, then in 10X SSC until blot set-up is ready. Make sure to notch the corner
of the nitrocellulose for orientation purposes and 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.
8. Neutralize gel in 0.5 M Tris/1.5 M NaCl (pH 7.0) for 30 minutes, rocking. Decant
the solution and repeat.
9. Rinse the gel in 10X SSC for 3 minutes, rocking.
10. 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.
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.
11. Assembling the Southern Blot:
-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.
-Place three pieces of Whatman 3M filter paper on top of wick.
-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**
-Place three more pieces of Whatman 3M filter paper on top of the
nitrocellulose.
-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).
-Wrap the whole set up in plastic wrap to provide stability to the stack
and minimize evaporation.
-Pressure should be applied to the top of the stack to enhance wicking
overnight.
16
(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 stereological pipette, add 50ml
2XSSC to the bag to hydrate your blot. 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 (2mg/mL) in
100°C hot block for 10 minutes followed by plunging into ice water.
5. For prehybridization of the nitrocellulose blot, add the 200µL of freshly
denatured Herring sperm to the prehybridization solution and mix.
6. Cut a corner of your bag and remove the 2X SSC. Pour in the prehybridization
solution using a stereological pipette. Reseal the corner 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 5L of the probe made on 1.9 and 200 L of
Herring sperm DNA by placing in 100°C hot block for 10 minutes. While in the
heat block, wrap tops of tubes in Parafilm to preserve the label in the next step.
9. Plunge into ethanol ice slurry for fast chilling, making sure not to erase all labels
written in marker. Just before use, add to the hybridization solution.
10. Remove the prehybridization solution from the bag by cutting a corner and
pouring off. Add the hybridization solution to the bag (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.
17
Following Day: 1.14 Detection of the DNA
**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 100ml 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 100ml 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 100ml 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 100ml 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.
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. Then decant off.
3. Dry blots between two Kimwipes.
4. Dry blots at 80o C for 15 mins.
5. Wash the blots in freshly made strep-avidin alkaline phosphatase (SA-AP)
conjugate for 25 minutes at room temperature. 1µL SA-AP per 1mL Buffer 2.
(Add only enough SA-AP conjugate to cover the blots (~10ml). Use gentle
agitation and occasionally pipette SA-AP over the blots.)
6. Decant and save the SA-AP in a 15ml tube. Save for step #8.
Wash the blot and test spot in Buffer 1 using 20 to 40-fold greater volume than
employed in step 3. Gently agitate blot for 15 minutes in Buffer 1.
(if you used 10 ml diluted SA-AP conjugate in step 3, wash with at least 200400ml Buffer 1.) Decant Buffer 1 into the sink.
7. Wash the blots for 10 minutes in Buffer 3, rocking. Decant Buffer 3 into sink.
(Do steps 8 and 9 at the same time and monitor the rate of color development.
The tube of saved SA-AP acts as a positive control.)
8. Add 1ml NBT/BCIP solution to the saved SA-AP. A blue color should develop
overtime. Wear gloves when working with NBT-BCIP.
18
9. Add 9 ml of NBT-BCIP solution to the blots. Allow the blots to develop for 15
minutes to 1 hour. Agitate the Tupperware.
10. 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 overdevelopment does not occur.
11. Once bands have developed, decant the NBT-BCIP solution in the appropriate
waste container and wash the blot in TE. This will terminate the color
development reaction. The TE can then be decanted into the sink.
12. Let the blots dry on a large Kimwipe. Then wrap in plastic wrap and label. The
lab tech will photograph and distribute the blots for your notebooks.
13. Measure the photograph of the cracking gel, and compare the relative position
of the plasmid band to the results of the blot. Interpret your results.
14. Record the development time with BCIP/NBT
1.16 Designing Primers
5’-------------------------------------------------------------------------------------------------------3’
698 bp
GST
Partial DNA Sequence for GST (Read left to right, top to bottom):
5’…GTATTCATGTCCCCTATACTAGGTTATTGAAAATTAAGGGCCTTGT
310
GCAACCCACTCGACTTCTTTTGA……….ATCCTCCAAAATCGGATCTGGT
960
TCCGCGTGGATCCCCGGGAATTCATCGTGACTGACTA………….…………..3’
The glutathione S-transferase protein consists of 232 amino acids. The sequence—using
the one-letter abbreviation for each amino acid—is shown below.
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS
MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDR
LCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPL
QGWQATFGGGDHPPKSDLVPRGSPGIHRD
Using this information, design the primers to amplify the GST gene. Once you have
designed the primers, fill out the oligonucleotide request form. The primers will then be
made on a DNA synthesizer.
1.17 PCR
1. Set up 7 - 0.5 ml PCR reaction tubes according to the PCR chart in Appendix I.
Read the chart carefully and make sure you add the correct amounts of reagents.
PCR is a very sensitive reaction and adding the incorrect amounts of reagents
19
2.
3.
4.
5.
6.
7.
8.
9.
may cause poor results. Appropriately label your tubes with your group color
and tube number!
For Sample 4, pick up three individual colonies from your transformed plate with
a sterile toothpick and place into a 1.5 ml microfuge tube filled with 50 µL of
sterile dH2O. Take 3µL of this bacterial solution and use as your “template DNA”.
Do the same for Sample 5 using non-transformed bacteria.
Before mixing the reactants, calculate how much water must be added to make a
total of 25L (including the Taq Polymerase). This is necessary because the
amount of template DNA that you add might differ from tube to tube.
If you add too much DNA, nonspecific amplification may occur—ask your TA how
much DNA to add based on the approximate concentration of your plasmid
samples.
Add all reactants, except the Taq, while the tubes are on ice.
Lastly add the Taq polymerase.
Once all reactants are added to the tubes, 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.
Keep the tubes on ice until the entire class is ready to load the thermocycler.
The thermocycler will run for approximately 3hrs. After the 3hr. period is over,
the thermocycler will stay at a constant 4C until the tubes can be placed in the
refrigerator by the lab technician or TA. This will ensure that the PCR products
will not degrade.
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. Make 300 ml 1X TAE.
2. Prepare a 2% agarose gel. Note: The 2% agarose solution will solidify quickly!
Pour gel while still relatively hot.
3. Remove 20µL of PCR product from each tube; add to 4 µL of 6X sample buffer.
Store the remaining PCR product at 4°C.
4. Once your gel is set, remove the comb and place the gel in the running box.
Cover the gel with 1X TAE buffer.
5. Load your DNA samples and 10µL of 100bp ladder into the gel.
6. Run gel at 100 volts for 1 hour.
7. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
8. Destain, examine, and photograph gel.
20
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
21
Module 2
RNA Interference
Introduction
In this module, a specific target gene product of Paramecium tetraurelia is depleted
using an RNAi feeding method. Paramecia are fed with an RNAase III-deficient E. coli
strain, HT115 bacteria transformed with a gene specific RNAi construct. The RNAi
construct has T7 RNA polymerase promoters on either side of the cloned gene which
expresses double stranded RNA upon IPTG induction. After 48 hours of feeding at 28°C
the level of target endogenous mRNA is determined by semi-quantitative Reverse
Transcriptase- Polymerase Chain Reaction (RT-PCR).
The lab tech has transformed E. coli HT115 cells. The “test” cells contain a plasmid with
the PAWN A gene, while the “control” cells contain a plasmid without the PAWN A gene.
Both the test and control cells are resistant to ampicillin and tetracycline.
2.1 Design Primers and PCR Protocol___________________________
5’-------------------------------------------------------------------------------------------------------3’
641 bp – PAWN A Sequence
ATGTATTTAT TAATTTTAAG TATATTGTAA TTTGGCATCG TGATTTAAGC 50
TCAAGAGACA AACAATACTG AAGAAGAGAT TTCAGATTAT TGTGATGCAG 100
TTGCCAAAGA CACTTCTTTT AAGGTGAATG TAACAGTTTC AGATATTAAC 150
AATAAAAATT ATTGTGTTGA AGGTGGATCT CGTGTGGCTT TATTCGACAC 200
AATTTAACAA GAAGATCAAT ATGTGTATTT GTCTGAACAT TATTGTGCCA 250
ACTTATAACA TTATCCAATT ACTTGCGAAC AATATTATCA CGCTTCAGAA 300
TATGATAAAA AAGTAAGAAT TTAAATACTA TCAATTAGGC CAAAGCCAAC 350
TATAAGAAGC TCTATGAAGA TTATAAGGCC ACTGGAGTAC CCAACTCTGA 400
TTGTTTGGGT ATTGCAAGAT TCGTTTTCTG TGCTGAATAA TTCAAATATT 450
GCAGCACAGA TGATGGAAAT ACCGATTATG AAATCTGCAG TTTCTTATGT 500
GTCATTTGGC AAAATAGATG TCCTGATTAC AGTGATATTT ACGATCGTGT 550
22
TTGTGCTAAT GGAGGAGGAG AAAATGGAAG ATGCAGTTAT GCAATTAACT
600
ATACTTTTCT GTTGTTTTTC ATTCTATTTT TATTATATTG A
641
The intron is nucleotide 313 to 333 and it is highlighted.
RNAi Construct is underlined. 560 bp (21
580)
Using this information, design primers and test your primers for endogenous mRNA.
Once you have designed the primers, fill out the oligonucleotide request form. The
primers will then be made on a DNA synthesizer.
Using the primers you designed, calculate the annealing temperature. 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
# of Cycles
1
1
2.2 Run RNAi and Endogenous PCR____________________________
Run the PCR protocols you designed with the primers you designed on genomic
Paramecium tetraurelia DNA. Use the following table to set up your PCR.
For each primer set you will run four different reactions.
Components
Template DNA (~100ng)
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
25mM MgCl2
0 µL
1.5 µL
3 µL
4.5 µL
10mM dNTPs
0.5 µL
0.5 µL
0.5 µL
0.5 µL
20µM F primer
0.5 µL
0.5 µL
0.5 µL
0.5 µL
20µM 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
23
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. Make 300 mL 1X TAE.
2. Prepare a 2% agarose gel using a 12 well comb. Note: The 2% agarose solution
will solidify quickly! Pour gel while still relatively hot.
3. Remove 10 µL of PCR product from each tube; add to 2 µL of 6X sample buffer.
Store the remaining PCR product at 4°C.
4. Once your gel is set, remove the comb and place the gel in the running box.
Cover the gel with 1X TAE buffer.
5. Load your DNA samples and 10 µL of 100bp ladder into the gel.
6. Run gel at 100 volts for 1 hour.
7. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
8. Destain, examine, and photograph gel.
2.4 Optimize PCR Protocol_____________________________________
After discussing the results with your instructors, optimize your PCR protocol.
2.5 Agarose Gel of Optimized PCR Protocol______________________
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. Make 300 mL 1X TAE.
2. Prepare a 1.5% agarose gel. Note: The 1.5% agarose solution will solidify
quickly! Pour gel while still relatively hot.
3. Remove 20 µL of PCR product from each tube; add to 4 µL of 6X sample buffer.
Store the remaining PCR product at 4°C.
4. Once your gel is set, remove the comb and place the gel in the running box.
Cover the gel with 1X TAE buffer.
5. Load your DNA samples and 10 µL of 100bp ladder into the gel.
6. Run gel at 100 volts for 1 hour.
7. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
8. Destain, examine, and photograph gel.
2.6 ON Bacterial Culture (Done for you)_________________________
The lab tech will start ON cultures from freshly transformed test and control HT115
cells. The cells will grow with shaking at 37C overnight.
24
2.7 Induce Bacteria and Feed to Paramecium_____________________
Part A: Inducing bacteria
1. Five hours before lab, the lab tech will add 1 mL of the test and control cells
previously grown ON to 50 mL LB amp medium.
2. The cells will shake at 37C for two hours.
3. After two hours of shaking measure the OD value of the culture at 595 nm using
the spectrophotometer. Use LB as the Blank. When the OD value reaches
between 0.3 and 0.4, proceed to the next step.
4. Add 125 µL 0.2M IPTG to each 50 mL bacterial culture. Shake at 37°C for 3
hours.
Part B: Purging Paramecium Cells
(Your TA will demonstrate procedure in MLS 224)
1. Obtain a 100 mL flask of paramecia grown in bacterialized wheat culture from
the 28C incubator. (These cells were added to the bacterialized 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 centrifuge tube against a centrifuge tube filled with water and
centrifuge at ¾ speed for 2 minutes in the IEC HN-SII centrifuge.
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 cells from the
bottom of the pear-shaped to the Dryl’s solution using a glass Pasteur pipette.
6. Keep the cells in the Dryl’s solution until you are ready to add them to the flasks
containing your test and control bacterial cultures.
Part C: Preparing the bacterial cultures for the paramecia
1. After three hours of shaking, transfer the test and control bacterial cultures to
large, sterile centrifuge bottles and harvest the bacteria by centrifuging at 5000
rpm, 4°C for 10 minutes in the Beckman J2-21 (JA-14 rotor).
2. Decant the supernatant broth into the waste jar.
3. Resuspend each bacterial pellet in 100 mL wheat culture containing 100 µL Amp,
100 µL Stigmasterol, and 250 µL 0.2M IPTG. Transfer resuspended pellet to flask.
4. Transfer approximately 1 mL of paramecia in Dryl’s solution to a depression slide
under a dissecting microscope. Using a Pasture pipette count and transfer 350
paramecia to each flask of culture.
5. Place flasks in 28C incubator.
2.8 Induce paramecium culture (Done for you)___________________
After 24 hours of incubation, your TA will add 100 µL Stigmasterol and 250 µL 0.2M IPTG
to each of the cultures and continue to incubate at 28C.
25
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 2Mercaptoethanol) in the appropriate waste container. Do not inhale fumes.
Induce Paramecium culture (Done for you)
After 48 hours of incubation, your TA will add 100 µL Stigmasterol and 250 µL 50mM
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 cells. 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. Transfer cells to 500 µL of Resting Solution in another depression slide. Try not to
transfer much culture medium.
3. Wait 10 minutes.
4. In the meantime, take 500 µL of 1mM BaCl2 solution to another depression slide.
Label the depression slide Test and Control.
5. After 10 minutes, transfer 1 cell at a time from the resting solution to the BaCl2
solution. Note the swimming behavior of each. First do this with control cells, then
with test cells.
6. Check at least 15 cells. – (Each person do 10 Control, 10 Test. 20 Total #’s.)
Part B: Filtering and Washing the Paramecium
(Your TA will demonstrate procedure in MLS 224)
1. Filter cells through a folded square of small Kimwipe into a 100 mL pear-shaped
centrifuge tube
2. Balance centrifuge tubes and centrifuge at ¾ speed for 2 minutes in the IEC HNSII centrifuge. Balance with LB.
3. Using a glass Pasteur pipettes remove the cells collected at the bottom of the
tube. (Your TA will demonstrate how to use circular motion to aid in removing
concentrated 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 empty pear-shaped centrifuge
tube and pipette in the cells to mix.
5. Balance and centrifuge the tubes at ¾ speed for 2 minutes in the IEC HN-SII
centrifuge.
26
6. Pipette out cells using the demonstrated technique and transfer to a 1.5 mL
microcentrifuge tube. 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 and remove the supernatant using a pipette.
Transfer approximately 100 µL of the tightly packed cells to a 0.5 mL centrifuge
tube.
3. Add 350 L of the lysis buffer (RA1) to the 0.5 mL tube of cells
4. Next, add 3.5 L of β-Mercaptoethanol. Use β-Mercaptoethanol in the hood
and place all pipette tips that come into contact with β-Mercaptoethanol into
designated waste container!
5. Use a 1 mL syringe to mix the solution approximately 3 times.
6. Place a pink filter column inside a collecting tube and pipette the cell solution
onto the center of the filter. Label both the collecting tube and the filter.
7. Centrifuge for 1 minute at 12,000 rpm
8. Remove filter and discard
9. Add 350 L of 70% ethanol to the sample in the collecting tube. Pipette up and
down to mix
10. Set up a blue spin column with collecting tube and label both.
11. Transfer the sample to the blue spin column and centrifuge for 1 minute at 8,000
rpm
12. Place column into a new collecting tube and label. Discard supernatant and used
collecting tube.
13. Add 350 L Membrane Desalting Buffer (MDB) and centrifuge at 12,000 rpm for
1 minute
14. Add 90 L of DNase Reaction Buffer and 10 L of DNase I stock solution to a
fresh 0.5 mL microcentrifuge tube.
15. Take 95 L of the DNase Reaction Mixture and add it to the center of the blue
spin column. Do not contact column and make sure DNase is absorbed. Let sit
at room temperature for 45 minutes.
16. Add 200 L RA2 Buffer to the spin column and centrifuge for 1 minute at 8,000
rpm. (If the tip of the column outlet comes into contact with the flow-through
for any reason, discard the flow-through and centrifuge again at 12,000 rpm for
1 minute.)
17. Place spin column into a new collecting tube
18. Add 600 L of RA3 Buffer to spin column and centrifuge for 1 minute at 8,000
rpm
19. Discard the flow-through and place the column back into the collecting tube.
27
20. Add 250 L RA3 Buffer to the spin column and centrifuge for 2 minutes at 12,000
rpm.
21. Place the spin column into the supplied RNase-free 1.5 mL microcentrifuge tube
and label.
22. Elute the RNA by adding 20 L of RNase-free water directly into the center of the
spin column. Centrifuge for 1 minute at 12,000 rpm
23. Take the liquid collected in the collecting tube and transfer to the top of column
to elute again. Wait 1 minute and then centrifuge at 12,000 rpm for 1 minute.
24. Discard spin column and aliquot 2 µL to a 0.5 mL tube for the lab tech to
nanodrop.
25. Place the tube 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 ug per tube using the RNA
concentration provided by the TA.
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. Tube 3 is the negative
control which will contain test RNA and no SuperScript III.
Reagents (µL)
RNA * 5 µg
Water *
50 µM Oligo dT
10 mM dNTPs
Total volume
Tube 1 (Test)
1
1
13
Tube 2 (+ Control)
1
1
13
Tube 3 (- Control)
1
1
13
*Your TA will inform you on how much RNA to add to each tube based on the results
from the Nanodrop. Calculate the volume of water that you need based on the amount
of RNA added. Add this chart to your lab notebooks with calculated volumes.
28
4. Incubate at 65C for 5 minutes then put on ice and add:
Reagents (µL)
5X First-Strand Buffer
0.1 M DTT
RNase Out
SuperScript III
Water
Total Volume
Tube 1 (Test)
4
1
1
1
0
20
Tube 2 (+ Control)
4
1
1
1
0
20
Tube 3 (- Control)
4
1
1
0
1
20
5. Place tubes in thermocycler and set to the following conditions:
cDNA synthesis Conditions
5 min
25C
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
29
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 to determine if there are
equal concentrations of mRNA in both the Test and the +Control.
Calmodulin primers:
Forward: 5’ CTG AAG CTG AAC TTC AAG 3’
Reverse: 5’ TCA TTT AGA AAC CAT CAT TCT 3’
Product length: 330-350 bp
1. Set up and label 9, 0.5 mL tubes according to the table below.
Calmodulin PCR (Note the endogenous set up is different)
Tube Treatment
µL of cDNA
µL of Master Mix
1
1X Test
1
49
2
1:10 Test
1
49
3
1:100 Test
1
49
4
1:500 Test
1
49
5
1X +Control
1
49
6
1:10 +Control
1
49
7
1:100 +Control
1
49
8
1:500 +Control
1
49
9
1X -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:
Sterile dH2O
375 µL
10X PCR buffer
50 µL
25mM MgCl2
30 µL
10 mM dNTPs
10µL
Calmodulin F primer
10 µL
Calmodulin R primer
10 µL
Taq Polymerase
5 µL
3.
4.
5.
6.
Mix the solution well by pipetteing up and down.
Add 49 µL of master mix to each labeled tube.
Add 1 µL of respective cDNA dilution to each tube. Mix well.
Spin tubes in microcentrifuge for 10 seconds.
30
7. Place tubes in PCR machine 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
Part C: Endogenous gene PCR
The endogenous PCR will allow us to check the mRNA levels and see if the gene was
down regulated due to RNA interference.
1. Set up and label 9, 0.5 mL tubes according to the table below.
Endogenous PCR
Tube Treatment
1
1X Test
2
1:10 Test
3
1:50 Test
4
1:100 Test
5
1X +Control
6
1:10 +Control
7
1:50 +Control
8
1:100 +Control
9
1X -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
172.5 µL (group dependent)
10X PCR buffer
25 µL
25mM MgCl2
20 µL (group dependent)
10 mM dNTPs
5 µL
Endogenous F primer
5 µL
Endogenous R primer
15 µL
Taq Polymerase
2.5 µL
31
3.
4.
5.
6.
Mix the solution well by pipetteing up and down.
Add 24.5 µL of master mix to each labeled tube.
Add 0.5 µL of respective cDNA dilution to each tube. Mix well.
Place tubes in PCR machine 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.
7. Run PCR with the conditions you determined during 2.4:
Endogenous PCR Conditions
Temp
Time (min)
Cycle
Initial Denature
30X
HOLD
Final Elongation
****************
8. 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 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. Make 300 mL 1X TAE.
2. Prepare a 2% agarose gel with a 12-well comb. Note: The 2% agarose solution
will solidify quickly! Pour gel while still relatively hot.
3. Remove 10 µL of PCR product from each tube; add to 2 µL of sample buffer.
Store the remaining PCR product at 4ºC.
4. Obtain an aliquot of 100bp ladder from the Lab Tech; this will be loaded directly
on the gel.
5. Once your gel is set, remove the comb and place the gel in the running box.
6. Cover the gel with 1X TAE buffer and load your samples.
7. Run gel at 120 volts for 45 minutes to 1 hour.
8. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
9. Destain, examine, and photograph your gel.
32
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. Make 300 mL 1X TAE.
2. 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.
3. Remove 10 µL of PCR product from each tube; add to 2 µL of sample buffer.
Store the remaining PCR product at 4°C.
4. Obtain an aliquot of 100bp ladder from the Lab Tech; this will be loaded directly
on the gel.
5. Once your gel is set, remove the comb and place the gel in the running box.
6. Cover the gel with 1X TAE buffer and load your samples.
7. Run gel at 120 volts for 45 minutes to 1 hour.
8. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium
bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.
9. 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.
33
Module 3
Proteomics
Introduction
In this module, you will compare the proteins found in wild type and mutant Paramecia
cilia. You will run a one dimensional polyacrylamide gel, cut out the bands of interest,
and compare the proteins present in those bands using mass spectrometry.
3.0 Preparation of Cilia (Done for you)
_____
Your TA will prepare the cilia from Paramecium using the following protocol:
1. Filter cell culture by slowly pouring paramecium cell culture through a funnel
lined with cheese cloth sandwiched between 2 large kimwipes into a clean 2800
mL flask.
2. Centrifuge cell culture to concentrate the cells.
3. Pipette cells into a beaker containing 100 mL of room temperature Dryl’s
solution. Mix the solution and then pour through a Kimwipe in a funnel into a
clean pear-shaped centrifuge tube . Centrifuge for 2 minutes at ¾ speed.
Remove trichocysts (fluffy layer on top of pellet) and put in waste container.
Transfer cells to clean Dryl’s buffer. Mix and centrifuge again. Do for a total of 3
washes.
4. Add cells to a flask containing 40 mL cold Dryl’s solution, then add 40 mL cold
STEN buffer. Keep on ice for 10 minutes. The cells should be immobilized within
10 minutes.
5. 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.
6. Transfer cell solution to pear-shaped flasks and centrifuge for 2 minutes on full
speed. Pour off supernatant into clean pear-shaped flask, leaving cell bodies in
old flask. Spin supernatant again for 2 minutes at full speed.
7. Transfer supernatant to 30 mL Cortex tubes with rubber sleeves. Spin in
Beckman J2-21 Centrifuge (JA-17 rotor) at 14,500 rpm for 20 minutes.
8. Add 3 mL of 10mM 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.
9. 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 5ml.
10. Place sample in a fresh 15ml Corex tube and wash the last tube with 1-2 ml Tris
EDTA pH 8.3.
11. Spin at 19,500 rpm in JA-20 rotor for 30 minutes.
12. After the spin, pour off the supernatant and resuspend the pellet in 400 - 600 µL
of 10mM Tris (pH 8.0). Be sure the suspension is homogenous.
13. Store at -80°C for future use.
34
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 Pawn A 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 J.
2. 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.
3. Carefully place 200 to 300 µL of your sample on top of the sucrose gradient.
Rinse the empty protein sample tube with 100ul of 10mM Tris (pH 8.0) and add
to gradient.
4. Balance the ultracentrifuge tubes in their buckets with 10mM Tris (pH 8.0). Note
the number on the bucket that corresponds to your sample.
5. YOUR TA WILL HELP YOU SET UP THE ULTRACENTRIFUGE ROTOR AND PLACE THE
ROTOR IN THE CENTRIFUGE!!
6. Ultracentrifuge (Sw60Ti rotor) at 45,000 rpm at 4°C for 1.5 hours.
7. After centrifugation there should be three different samples at the different
interfaces. See Appendix K. We are only interested in collecting the pure ciliary
membrane which is between the 20% and 45% sucrose gradients.
8. 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
gradients. Place the collected sample into a fresh 15ml corex tube.
9. Add 12 mL of 10mM Tris (pH 8.0) to each tube and invert to mix well.
10. Balance the tubes in their rubber sleeves.
11. Centrifuge in the Beckman J2-21 (JA- 17 rotor) at 17,000 rpm at 4°C for 30
minutes.
12. 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.
13. Resuspend the pellet in 100 µL of Membrane Buffer with protease inhibitors.
14. Remove 15 µL and place in a labeled microfuge tube for the Pierce Protein Assay.
Store the tube at -80°C.
15. Store remaining samples at -80°C.
35
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.
1.
2.
3.
4.
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 diagram in Appendix L). 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 comb. Once the line is drawn, remove the comb.
Test to see if the apparatus is leak-proof. Squirt 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 Kimwipe to
remove water droplets from between the plates of glass.
Before you pour your gel, have the TA or lab tech check your apparatus!
5. 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
3.29 mL
4X Resolving Buffer pH 8.9
2.60 mL
30% Acrylamide stock
4.00 mL
TEMED
10 L
Last:
Fresh 10% Ammonium persulfate 100 L
6. Gently swirl the solutions to mix WELL.
7. Using a Pasteur pipette, pour the gel by allowing the acrylamide 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.
8. Overlay the acrylamide with dH2O. Do this by gently adding the dH2O with a
glass Pasteur pipette. You will be able to see a distinct line between the
dH2O and the resolving gel solution.
36
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).
10. Once your gel has polymerized, wrap the gel/casting apparatus in a damp
paper towel and then plastic wrap. Label appropriately. Store in the cold
room.
9.
3.3 Pierce Protein Assay
Pierce Protein Assay (See directions in Appendix M)
Use the chart provided to develop a standard curve using BSA standards and to
determine protein concentration.
1. Dilute protein sample: Make 10X and 20X dilutions for each protein sample. For
example, to make a 20X dilution, add 5 L of your sample to 95 L of sterile
dH2O. For a 10X dilution add 10 L of your sample to 90 L of sterile dH2O.
2. Make dye solution: Use Solutions A and B from the Pierce Protein Assay Kit.
They should be mixed 50:1…but make up only the amount you will need. Mix
the dye in a 50 mL tube.
3. Add BSA and dH2O according to the directions in Appendix M.
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.
7. Place your standard into a clean cuvette.
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.
8. Read OD at 562 nm.
9. Repeat for each standard and sample, including duplicates.
If only using 1 cuvette, make sure to rinse with dH2O between each standard.
10. Once the standards are complete, read your samples. You should blank the
instrument with dH2O and dye. If using only 1 cuvette, make sure to rinse with
dH2O between each sample.
11. Find the average of each standard and sample duplicates.
12. Establish a standard curve using the OD values obtained with your BSA
standards: graph Concentration (x-axis) vs. OD (y-axis) on graph paper. Using
this graph, calculate the protein concentrations in your three samples.
13. Use Excel to plot your data on a second chart (This is homework). Make sure to
paste your Excel chart in your notebook properly labeled.
37
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. Mix the following components in a 15 mL tube.
3. Sterile dH2O
6.10 mL
4X Stacking Buffer pH 6.8
2.50 mL
30% Acrylamide stock
1.30 mL
TEMED
10 L
Last:
Fresh 10% Ammonium persulfate 50 L
4. Before adding the ammonium persulfate, pour the dH2O off the resolving gel and
dry with a Kimwipe.
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. If it overflows when inserting the comb this is okay. Clean the
comb thoroughly with ethanol before inserting.
7. Being careful to avoid making air bubbles, insert the clean comb until there is no
air between the wells. 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.
8. Allow 30 minutes for the gel to polymerize. Thaw your protein samples on ice
while waiting.
9. Once the gels are set, remove them from the casting stand and assemble in the
gel box (See Appendix N). Note: Do not remove the comb yet.
10. 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.
11. Add 1X PAGE Running buffer to the lower chamber until the appropriate level for
the number of gels in the box is reached.
38
12. Carefully remove the comb.
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. Each gel will have test and control sample lanes
and a protein marker. Your TA will help you to equalize the concentrations of test and
control so that the lanes on the gel are comparable . Any empty lanes should be filled
with sample buffer.
1. Calculate the volumes of sample, 10X SDS sample buffer, and water needed for
each tube:
-Each tube needs 60 µg of protein total. Using the concentrations you
calculated off of your standard curve, calculate how many microliters
equals 60 µg.
2. Add the calculated volumes of water, protein, and 10X SDS sample buffer to a
1.5 mL tube. Place the remaining protein samples in the freezer.
3. Add 1 µL of β-Mercaptoethanol to protein sample mixture.
4. Boil samples for 5 minutes right before you are ready to load. After boiling, keep
samples on ice while loading gel.
5. Using gel loading tips, load 20 μL of each of your samples into the gel in the
following order:
Lane 1, 3, 4, 6, 7, 9 and 10: SDS sample buffer
Lane 2: Prestained protein marker
Lane 5: 60 µg WT
Lane 8: 60 µg PwA
6. Run the gel at 50 mA for 1-1 ½ hours.
7. 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.
8. Put the gel into a plastic container and cover with Coomassie stain. Stain
overnight.
9. Pour off stain into original container.
10. Add destain to gel. Wash and discard destain in correct waste container.
11. Rinse gel with destain again and decant destain into correct waste container.
12. Submerge the gel in more destain. Rock gently overnight.
13. The Lab tech will save these gels in destain until the next lab.
39
3.5 Cutting out gel bands and trypsinizing proteins in preparation_ for
Mass Spectrometry_________________________________________
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.
1. Clean a 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 gel from its container and place on the glass plate.
3. Label a 1.5 mL microcentrifuge tube with appropriate identification for each
protein band to be analyzed. Select the razor blade and clean with 95% ethanol
before using and between cutting each band.
4. Select bands of interest based on visible differences in intensity of Coomassie
staining. Cut out the selected gel bands into small pieces (1mm) and transfer
them to the labeled tubes carefully.
5. Add 900 µL of HPLC-grade water to each tube. Incubate at room temperature
for five minutes.
6. 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 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.
7. Add 750 µL destain solution (50mM 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.
8. 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.
9. 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.
10. 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 2 minutes at room temperature.
40
11. 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.
12. Open lids and place tubes in Speed Vac for 5 minutes to dry samples completely.
Close the lids before proceeding to the next step.
13. Place the tubes on ice for 5 minutes. Add 25 µL ice cold trypsin/50 mM
ammonium bicarbonate solution to each tube. Incubate on ice for five minutes.
Add 25 µL cold 50 mM ammonium bicarbonate solution to each tube. Make sure
the gel is completely immersed in the solution. Add more ammonium
bicarbonate solution if necessary.
14. Incubate the tubes on ice for an additional 30 minutes.
15. Transfer the tubes to a 37°C incubator. Incubate the tubes overnight (8-16 hrs).
The Next Day:
16. 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. (If it is too difficult to
remove the liquid because the gel pieces are too big, wait until the next step to
pull off the peptide solution.)
17. Add 150 µL of 50% Acetonitrile / 2.5% formic acid to the tubes of gel slices and
vortex.
18. Spin these tubes for 5 minutes at full speed and transfer the peptide solution
(supernatant) to their respective 0.5 mL sample tubes.
19. Add 100 µL of 100% Acetonitrile to the tubes of gel slices and vortex.
20. 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.
21. Dry the samples using the Speed Vac for 2 to 4 hours.
22. 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.
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.
41
Appendix A: Solutions Guide
GENERAL SOLUTIONS
Ampicillin Stock
100 mg/ml stock solution: for example, 0.5g ampicillin sodium salt into 5 ml
dH2O. Filter sterilize, and store at -20°C.
LB amp
100 µg/ml final concentration: 1:1,000 dilution of ampicillin stock into LB broth.
When making LB amp plates, add 1 ml ampicillin stock (100mg/ml) into 1L LB
Agar broth. *NOTE: Ampicillin is heat-sensitive, so LB agar broth must be cooled
to 60°C after coming out of the autoclave, before the ampicillin is added. Setting
the water bath to 60°C and letting the LB agar broth cool in there for an hour is a
good way to ensure the LB agar doesn’t solidify.
When making LB amp broth, add 1ul of ampicillin stock (100mg/ml) for each 1ml
of LB broth.
TE buffer
Need (final conc.): 10mM Tris-Cl (pH 7.5), 1mM EDTA (pH 8.0)
Make from liquid stocks of Tris-Cl and EDTA
5ml 2M Tris-Cl (pH 7.5)
2ml 0.5M EDTA (pH 8)
993ml dH2O
2M Tris-Cl (pH 8.0)
177.6g Tris-Cl
10.6g Tris-base
In ~950mL sterile dH2O
**pH 8.0**
Bring up to 1L with sterile dH2O
0.5M EDTA (pH 8)
18.6g EDTA disodium salt (FW= 372.2)
In ~75ml sterile dH2O
Heat in microwave to dissolve salt
***bring pH to 8.0***
Bring up to 100mL with sterile dH2O
42
50X TAE stock (pH 8.5)
242g Tris Base (FW= 121.14)
In ~700ml sterile dH2O
Carefully add 57.1mL Glacial Acetic Acid
100mL 0.5M EDTA (pH 8.0)
Bring up to 1L with sterile dH2O
pH 8.5, but no adjustment needed
*Dilute 50X TAE stock 1:10 for a 5X stock*
6X DNA sample buffer
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 sample buffer
20X SSC (pH 7)
175.3 g NaCl
88.25 g Na3 Citrate•2H2O
1 L dH2O
**pH 7.0**
2X SSC
10ml 20X SSC
90ml dH2O
10X TBS (pH 7.4)
25.1g Tris HCl
4.8 g Tris Base
80 g NaCl
800mls of H2O.
Make volume up to 1 L with high purity distilled or deionized water.
Once prepared, TBS is stable at 4°C for 3 months.
43
10X TBS-T
10X TBS, 1% Tween-20
10 ml Tween-20 (use large orifice tips to pick up Tween)
1L 10X TBS
Phosphate Buffer (PBS)
4.0g NaCl
0.1g KCl
0.72g Na2HPO4
0.12g KH2PO4
In ~400ml sterile dH2O
**pH 7.4**
Bring to 500ml with sterile dH2O
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 50ml
**add 5 mg/ml lysozyme just before use**
Solution II
2 ml 1M NaOH
1 ml 10% SDS
7 ml dH2O
**Prep fresh**
5M Potassium Acetate
29.5 ml glacial Acetic Acid
100 ml dH2O
Add KOH pellets until pH=4.8
Store in refrigerator
44
Heat-treated RNase A (100mg/ml)
Dissolve 100 mg (0.1g) of pancreatic RNase A in 1 ml 10mM Tris-Cl/15mM 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
100ml dH2O
Salt Saturated 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
1.4: Transformation
50 mM CaCl2
0.73 g CaCl2
100 ml dH2O
** Make fresh**
Transformation buffer
1ml 100 mM CaCl2
1 ml 100 mM Tris (pH 8.0)
1 ml 100 mM NaCl
7 ml dH2O
**Store at 4°C**
45
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**
1.11: Southern blot
Cracking Buffer
See 1.8 above
0.5M NaOH/0.8M NaCl
20 g NaOH
46.752 g NaCl
1 L dH2O
0.5M Tris/1.5M NaCl (pH 7)
250 ml 2 M Tris-base solution (pH 8)
87.6 g NaCl
750 ml dH2O
** pH 7**
46
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 1M 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 1M 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)
10ml sterile dH2O
**Doesn’t keep more than 24 hours**
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
47
1.15: Development of Blot
Buffer 1 : Final Concentration: 0.1 M Tris-Cl
0.15 M NaCl
8.7 g NaCl
15.764 g Tris-Cl
1 L dH2O
Buffer 2: 3% (w/v) BSA in Buffer 1
3g 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: 0.1M Tris-Cl pH=9.5
0.1M NaCl
50 mM MgCl2
15.764 g Tris-Cl
5.844 g NaCl
10.15 g MgCl2
1 L dH2O
1.17: PCR
20 µM Primer Dilutions (from 500 µM stock primers)
5 µL 500µM stock
120µL 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)
500µL dCTP
500µL dTTP
500µL dATP
500µL dGTP
3 ml dH2O
*1 µL of 10mM dNTP mix per 50µL reaction*
48
MODULE 2
2.2 PCR
20 µM Primer Dilutions (from 500 µM stock primers)
5 µL 500µM 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 10mM dNTP mix per 50 µL reaction*
2.7 Induce Bacteria and Feed to Paramecium
0.2 M IPTG
750 µL dH2O
37.5mg IPTG
Wheat culture
Wheat grass tea buffered with:
3.75mM Na2HPO4+7H2O
3 mg/L Stigmasterol
Dryl’s solution
1mM Na2HPO4
1mM NaH2PO4
1.5mM CaCl2
2mM Sodium Citrate
pH 6.8
Stigmasterol
5 mg/mL dissolved in 100% ethanol
49
2.9 Harvest cells and Isolate RNA
Resting Solution (5.1.1)
1mM Citric acid
1mM Calcium hydroxide
1mM Tris base
5mM KCl
pH 7.04
5mM BaCl2
1mM Citric acid
1mM Calcium hydroxide
1mM Tris base
5mM BaCl2
pH 7.09
2.10 Making cDNA
50uM Oligo dT
20µM Primer
5’ CGGCTCGAGTTTTTTTTTTTTTTTTTTTT 3’
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
2.11 Template Control and Endogenous RT-PCR
20 µM Primer Dilutions (from 500 µM stock primers)
5 µL 500µM stock
120 µL dH2O
**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in
smaller batches**
50
MODULE 3
3.0 Preparation of Cilia
Dryl’s solution
1mM Na2HPO4
1mM Na2H2PO4
1.5mM CaCl2
2mM Sodium Citrate
pH 6.8
STEN Buffer
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
180mM KCl
60mM CaCl2
Membrane Buffer
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 1mg/mL
Store at -20C in 1mL aliquots
*Stable for 6 months*
51
Pepstatin A
Dissolve in methanol 1 mg/mL
(5mL methanol into 5 mg bottle and shake)
Store at -20C in 1mL aliquots
*Stable for 1 month*
100mM 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 (0.02g/ml)
*Add these immediately before use*
Tris EDTA
0.0121 g 1mM Tris Base
0.0034 g 0.1mM Na2EDTA
100 mL dH2O
*pH 8.3*
3.1 Ultracentrifugation
10mM Tris pH 8.0
5 mL 1M Tris, pH 8.0
495 mL dH2O
66% Sucrose
171 g Sucrose
90 mL 10mM Tris pH 8.0
Make the night before and place in shaker at 37o C overnight to dissolve
52
3.2 Preparation of Resolving Gel
4X Resolving Buffer (pH 8.9)
18.17g Tris base
10 g SDS (measure in hood)
100 mL dH2O
**pH 8.9**
3.4 SDS-PAGE
4X Stacking Buffer (pH 6.8)
6.055 g Tris base
0.4 g SDS
100 mL dH2O
**pH to 6.8**
10X SDS Sample Buffer
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*
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
200 mL Methanol
50 mL Glacial Acetic Acid
1 g Coomassie Blue
250 mL dH2O
53
Coomassie Blue Destain
200 mL Methanol
75 mL Glacial acetic Acid
725 mL dH2O
3.5 Band Cutting and Trypsinization of Gel Plugs
Trypsin in 50mM ammonium bicarbonate
Add the whole trypsin vial to 1.6 mL of 50mM Ammonium Bicarbonate
*make up same day and keep on ice
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, slants 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 nonsterile 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.
54
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.
3. Pass the spreader through the flame of a Bunsen burner to burn off the alcohol.
(This sterilizes the spreader).
***IMPORTANT***
Keep the dish of alcohol behind the Bunsen burner.
Keep the alcohol dish covered when you are not using it.
Keep your hand above the spreader at all times or flaming alcohol may roll toward
your hand.
If the dish of alcohol catches on fire, cover the dish with the glass lid and it will go out.
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.
Appendix D: Use of a Rainin Pipettor
Take note:
 Never rotate the volume adjustor beyond the upper or lower range of the
pipette man, as stated by the manufacturer.
 Never use the pipette man without the tip in place; this could ruin the precision
piston that measures the volume of fluid.
 Never lay down the pipette man 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 pipette man in fluid.
 Never flame pipette man tips.
If you drop your pipette man, the precision piston system can be damaged; therefore, if
your pipette man is dropped, be sure to check the pipetting accuracy has not been
affected.
55
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 pipette man 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 pipette man 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, repipette 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 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.
56
Appendix E: Pipette Exercises
Pipette Exercise #1
Determine and record the pipettor best suited for each of the measurements listed
below.
Add the indicated amounts to labeled microfuge tubes. Use the matrix below as a
checklist while adding solutions to each microfuge tube.
Tube A (green)
Tube B (red)
Tube C (blue)
Solution 1
10 µL
2 µL
598.6 µL
Solution 2
25 µL
0.015 mL
0.200 mL
Solution 3
0.0963 mL
183 µL
201.4 µL
Determine the total volume being added to each of the tubes. To check that your
measurements are accurate, set a pipettor to the final volume and carefully withdraw
the solution from each tube. Is the tip just filled? If measurements are inaccurate,
repeat the exercise to obtain a near-perfect result.
Pipette Exercise #2
Using the p1000 and the p200 pipettors, perform the following:
Set the pipettor to its maximum volume.
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 gm/mL or 1 g/L.
Therefore, you can determine the accuracy of your pipetting, e.g., 1000 L of water will
weigh 1gm. 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
10 L =_______mL
100 L =_______mL
1000 L =_______mL
0.001 L =________mL
0.11 L
=________mL
0.01 mL
=________mL
1L
=________mL
57
APPENDIX F: GST Plasmid Map
58
APPENDIX G: GST Plasmid Map
59
APPENDIX H: Frequently Used DNA/Protein Markers
Lambda DNA-Hind III Digest
100 bp DNA Ladder
Prestained Protein Marker
1Kb DNA Ladder
60
APPENDIX I: Streak Plate Method
1
2
3
4
5
Flame loop in between each step
except between 4 and 5.
Do not flame loop between steps 4
and 5.
61
APPENDIX J: PCR Reagents and Conditions for 1.17
Cycling Program: GST
94°C
94°C
50°C
72°C
5 min
1 min
1 min
1 min
Initial Elongation
94°C
51°C
72°C
1 min
1 min
1 min
25X
72°C
4°C
10 min
HOLD
Final elongation
***************
5X
SAMPLES
Initial Stock
Concentration
25mM
10mM
20µM
20µM
5U/µL
Components
1
2
3
5
3
colonies
5
6
7
1
5
4
3
colonies
5
Template DNA*
10X Buffer
1
5
1
5
1
5
1
5
MgCl2
dNTPs
Forward Primer
Reverse Primer
Taq Polymerase
0.5
1
1
1
0.5
1
1
1
1
0.5
3
1
1
1
0.5
3
1
1
1
0.5
3
1
1
1
0.5
3
1
XXX
1
0.5
3
1
1
XXX
0.5
dH2O
*NOTE: Samples 1-3, 6 & 7: Use PLASMID DNA
Sample 4: Transformed Colonies
Sample 5: Non-Transformed Colonies
DESIRED FINAL VOLUME: 50 µL
62
APPENDIX K: Sucrose Gradient Calculations
How to make __% Sucrose from 66% Sucrose
Add __ mL of
66% sucrose
Add __ mL 10mM
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
63
APPENDIX L: Sucrose Gradient Tubes after Ultracentrifugation
20%
Ciliary membrane
45%
Partial axoneme
55%
Pure axoneme
66%
64
APPENDIX M: Protein Gel Plate Setup
65
APPENDIX N: PIERCE PROTEIN ASSAY for Module 3
Label
S1
S2
S3
S4
S5
S6
S7
Label
L Alb
(Stock
2mg/mL)
0
2.5
5.0
7.5
10.0
12.5
15.0
L
Sample
L
dH2O
mL
Dye
[Alb
g/mL]
100
97.5
95.0
92.5
90.0
87.5
85.0
2
2
2
2
2
2
2
0
50
100
150
200
250
300
L
dH2O
mL
Dye
OD Values
OD Value
Series
Series
A
B
Average
OD Value
Average
g/mL
Original
Solution
g/mL in
Cuvette
2
2
2
2
Label
Average g/mL
Original Solution of
each treatment
g /L
Original solution
L of protein that
equates to 60 or
100 g total
WT
PwA
66
APPENDIX O: Protein Gel Running Setup
67
APPENDIX P: RPM to G-Force Conversions
Equipment
Beckman J2-21 w/JA 14 rotor (250mL tubes)
Beckman J2-21 w/JA 17 rotor (30 mL tubes)
Beckman J2-21 w/JA 17 rotor (30 mL tubes)
Beckman J2-21 w/JA 17 rotor (30 mL tubes)
Beckman J2-21 w/JA-17 rotor (15 mL tubes)
IEC Centra 7 Desktop w/ 15 mL tubes
IEC Centra 7 Desktop w/ 50 mL tubes
Eppendorf Centrifuge 5702 w/ 15 mL tubes
Eppendorf Centrifuge 5702 w/ 50 mL tubes
Eppendorf Centrifuge 5415 C w/ 1.5 mL tubes
Sorvall Legend Centrifuge w/ 1.5ml tubes
Ultracentrifuge
Ultracentrifuge
RPM
5,000
5,000
9,500
15,000
17,000
2,800
2,800
2,800
2,800
14,000
14,000
45,000
21,600
G-Force
(RCF)
3,840
3,440
12,400
31,000
39,000
1,098
1,098
1,120
1,180
15,980
18,800
208,000
45,000
68