Name: _________________________________________
Biology 309 Lab Notebook
This is a “guided lab notebook” for you to keep well-organized notes
about procedures and record experimental data for experiments as they
are performed. It is “guided” because, instead of providing a notebook
with blank pages, you are given a framework for recording certain key
information. What you are required to include is not the thorough record
that would be expected in an actual research laboratory, but will model
how such notes should be maintained.
Note keeping guidelines
Notes must be neat. If you can do so in ink, fine. But if not, then you must use a pencil. When
necessary continue not taking on back side of pages.
The first page of each section must be completed before coming to lab, and will be collected at
the start of lab. If you are working on it in lab it will not be accepted. Completing this page is
critical to your preparation for the lab exercise.
Other pages of the lab notebook will be collected “periodically”.
Recording Procedures: Each student is expected to take notes as procedures are performed.
Recording Data: In some labs, it will be easier for one student to record the data as the partner
carries out a procedure. This is fine, but the data should be copied over to the other lab notebook.
All graphs should be fully and properly formatted per the Figure preparation guidelines.
Graphing, calculations, and other data processing: must be done but each student individually;
although you are expected to “compare notes” to assure agreement after doing so. Other than to
transfer data, copying from another person’s notebook will be considered plagiarism.
Solution Preparation
It is expected that both group members participate in the preparation of all solutions.
Table of contents
Solution Preparation -- page 3
Histology
-- page 9
PET and Hill reaction -- page 13
Culturing B16 cells -- page 23
Protein measurement -- page 31
Gel Electrophoresis -- page 39
Immunoblotting
-- page 45
Page 1
Page 2
Name: _________________________________________
Solution Calculation and Preparation
1. What is the equation that is used when diluting a reagent solution to a lower concentration?
2. What is the equation that is used for calculating the grams of a dry reagent for a molar
concentration solution?
3. What is the equation used when calculating the grams of a dry reagent for a %(w/v) solution?
4. What is the equation used when calculating the volume of a liquid reagent for a %(v/v) solution?
For Hill Reaction lab
Homogenization buffer (50 ml) – Prepare before lab day
Components
Tricine-NaOH1
Final conc.
50 mM
MW
________
/ 50 ml
________ g
Sucrose
0.4 M
________
________ g
NaCl
10 mM
________
________ g
pH 7.8
1
The designation "-NaOH" after Tricine indicates that the pH of the solution should be adjusted
with NaOH.
Calculations
for Tricine:
for Sucrose
for NaCl:
Prepare by sequentially dissolving Tricine, Sucrose and NaCl in 40 ml of upH2O in a 100 ml
beaker on a magnetic stirrer. Adjust the pH with NaOH (first with 10M, and then with 1M).
Transfer to a 50ml graduated cylinder and bring volume up to 50 ml with upH2O. Store
solution in the refrigerator.
Page 3
For Histology lab
1. Isopropyl alcohol (IPA) solutions
solution
volume of IPA
Volume of H20
250 ml of 70% (v/v) IPA
___________ ml
___________ ml
50 ml of 85% (v/v) IPA
___________ ml
___________ ml
200 ml of 95% (v/v) IPA
___________ ml
___________ ml
For each solution, add the IPA to a graduated cylinder, bring volume up to the indicated final
volume with roH2O, and transfer to a reagent bottle
.
Calculations
for 70%:
for 85%:
for 95%:
2. Scott’s solution (100 ml)
Components
Final
conc.
MW
/ 100 ml
NaHCO3
240 mM
________
________
MgSO4
1.7 mM
________
________
Calculations
For NaHCO3:
For MgSO4:
Page 4
Name: _________________________________________
For Protein Measurement/Spectrophotometry lab
1. Homogenization buffer (100 ml) – Prepare before lab day
Reagents
Final Conc.
MW
/ 100 ml
Tris-HCl1
50 mM
________
________ g
20 mM EDTA•2Na
1 mM
NA
________ ml
pH 7.5
1
In powdered form Tris is sometimes labeled as "Trizma” or Trizma-base". The designation "HCl" after Tris indicates that the pH of the solution should be adjusted with HCl.
Calculations
for Tris:
for EDTA:
Prepare by dissolving Tris in 80 ml of upH2O in a 250 ml beaker on a magnetic stirrer. Then
pipet in the appropriate volume of EDTA Adjust the pH with HCl (first with 10M, and then
with 1M). Transfer to a 100ml graduated cylinder and bring volume up to 100 ml with upH2O.
Store in a 125 ml reagent bottle in the refrigerator.
2. BCA reagent (30 ml) – (you will not actually prepare this solution)
Components
BCA reagent A
4% CuSO4 (w/v)
Final conc.
100%
0.08%
/ 30 ml
30 ml
_______ml
Calculations
for CuSO4:
The BCA reagent is prepared by mixing the appropriate amount of 4% CuSO4 with Reagent A,
which contains a proprietary mixture of bicinchoninic acid and other chemicals.
Page 5
For Electrophoresis and Electroblotting lab (2 solutions)
1. Reservoir Buffer (500 ml) – Prepare before lab day
Components
Tris1
1
2
Final Conc.
25 mM
MW
________
/ 500 ml
________ g
Glycine
200 mM
________
________ g
SDS2
0.1% (w/v)
NA
________ g
In powdered form, Tris is labeled as "Trizma” or Trizma-base".
SDS is an abbreviation for sodium dodecyl sulfate (or sometime ‘sodium laurel sulfate’).
Calculations
for Tris:
for Glycine:
for SDS:
Prepare by sequentially dissolving Tris, Glycine and SDS in 400 ml of upH2O in a 1 L beaker on
a magnetic stirrer. The pH of this solution should not be adjusted, since the quantities of Tris and
glycine added result in an appropriate pH (8.3). You should check the pH and see that it falls
reasonably closed (±0.5 pH unit) to this value. Transfer to a 500 ml graduated cylinder, bring
volume up to 500 ml with upH2O, and store in a 500 ml reagent bottle.
2. Transfer buffer (Liter) – Prepare before lab day
Components
1
Final conc.
MW
/L
Tris 1
25 mM
________
________ g
Glycine
200 mM
________
________ g
Methanol
20 % (v/v)
NA
________ ml
In powdered form, Tris is labeled as "Trizma” or Trizma-base".
Calculations
for Tris:
for Glycine:
for Methanol:
Prepare by dissolving the Tris and glycine in 600 ml of upH2O in a 1L beaker on a magnetic
stirrer. Add the appropriate volume of methanol, and then bring solution up to its final volume. As
for the above solution, do not adjust the pH. Store this solution a 1L reagent bottle in the
refrigerator.
Page 6
Name: _________________________________________
3. Sample Denaturation Buffer (you will not actually prepare this solution)
Components
Final Conc.
Stacking gel buffer 40% v/v
MW
NA
/ 25 ml
________ ml
glycerol
10% w/v
NA
________ g
SDS
2% w/v
NA
________ g
0.1% w/v BPB1
0.01% w/v
NA
________ µl
βme2
6% v/v
NA
________ ml
1
= Brome-phenol-blue
2
= β-mercaptoethanol
Calculations
for Stacking gel buffer:
for Glycerol:
for SDS:
for BPB:
for Βme2:
For Immunoblotting Lab
1. Wash buffer (150 ml) - prepare before lab day
Components
Final conc.
MW
/ 150 ml
Tris-HCl pH 7.5
10 mM
________
________ g
NaCl (powder)
150 mM
________
________ g
NA
________ ml
1
1% Tween-20
0.05% v/v
1
Tween-20 is a mild detergent that reduces non-specific binding of antibodies to the nitrocellulose
membrane and contributes to membrane blocking.
Calculations
for Tris:
for NaCl
for Tween-20:
Prepare by dissolving Tris and NaCl in 120 ml of upH2O in a 250 ml beaker on a magnetic stirrer.
Then pipet in the appropriate volume of Tween-20. Adjust the pH with 1 M HCl. Transfer to a
250ml graduated cylinder and bring volume up to 150 ml with upH2O. Store in a 250 ml reagent
bottle.
Page 7
Page 8
Name: _________________________________________
Histology exercise
What is the purpose of this lab exercise?
Tissue preparation
Describe the tissues ressected from the mouse and how they were handled
Actual fixation, dehydration and infiltration schedule
Solution
Date & time-in
Date and time-out
Time length (hr)
Identify each of the tissue blocks that you prepare
Block #
Tissue
Orientation
Page 9
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Name: _________________________________________
Histology exercise, cont.
You are expected to track precisely the ribbons that you cut; tissue, thickness, orientation
Slide preparation
Slide #
Tissue
Thickness
Staining time
Hemotox.
Eosin
Describe the qualities of the two best slides (one for each tissue) you prepare
-- condition of tissue; distinct tissue structures you were able to identify
Slide # and tissue:
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Name: _________________________________________
Studying PET through the Hill Reaction
Answer questions A - D before coming to lab
A. What is the purpose of this exercise?
B. Why are chloroplasts used in this exercise instead of purified thylakoids?
C. What is the hypothesis stated in the lab exercise about the location where DCPIP absorbs
electrons?
D. Based upon this hypothesis, which of the inhibitors should block DCPIP reduction in the Hill
reaction? Explain your reasoning for each of the three inhibitors.
E. Why is it necessary to include measurements of chlorophyll concentration and light intensity in
the calculation of the Hill reaction?
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Name: _________________________________________
Studying PET through the Hill Reaction
Recording of protocols and data
For these protocols…
Chloroplast preparation
Chlorophyll measurement
Light measurement
Record each step of the procedures, focusing on specific volumes, quantities, or other
quantitative parameters, and stating the purpose for that step. Be sure to record critical values
such as the chlorophyll absorbance measurement and light intensity measurement.
Hill reaction assays:
Record general steps of the protocol, focusing on specific volumes, quantities, or other
quantitative parameters, and stating the purpose for that step. For all assays, record the
absorbance measurements made in tables as illustrated on the next page. Indicate how protocol
was varied for control and inhibitor assays.
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Name: _________________________________________
Studying PET through the Hill Reaction
Recording of Hill reaction data
Create tables like this to record your raw data
Assay Description:
Modifications, etc.:
Min
Abs
0
0.5
1
1.5
etc
______
______
______
______
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Name: _________________________________________
Studying PET through the Hill Reaction
Results calculations
Show calculations clearly and neatly is spaces provided
A. Using the Beer-Lambert law (abs = concentration x abs coef.), calculate the chlorophyll
concentration of your chloroplast suspension. The absorption coefficient for chlorophyll at 652
nm is 35.4 ml cm-1 mg-1.
ABS 652 = _______
dilution factor =_______
[chlorophyll] (and units) = ________
(value should be between 0 and 10.)
B. Following the instructions in the manual and using the above value, calculate the mg of
chlorophyll in the chloroplast suspension added to each Hill assay mixture:
________ mg chlorophyll in assay
C. Following the instructions in the manual, and the light intensity measured in lux, calculate the
photon fluence rate:
Measured light intensity: ________ lux
Photon fluence rate (and units) = _______
D. Determine the rate of DCPIP reduction
Slope 1 (and units) = ________
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Name: _________________________________________
Studying PET through the Hill Reaction
Results calculations, cont.
E. Following the instructions in the manual, complete the calculation of the Hill reaction rate.
Show calculations for each assay:
Rate 1: ___________ (correct calculations should yield a number between 0 and 1)
Average rate (and units): _______________
Control:
What was effect of eliminating light?
Inhibitors
What was the effect of adding each inhibitor?
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Name: _________________________________________
Culturing and Fluorescent Staining of B16 melanoma cells
Week 1 - Culturing and Passaging of cells
Answer questions A - E before coming to lab
A. What is the purpose of this lab exercise?
B. Is your B16 cell culture a primary or secondary culture? Explain.
C. Why is it necessary to passage cultured cells periodically; explain all the reasons why this is
necessary.
D. What are the 3 components of CDMEM and what are their functions?
E. Why is Trypsin/EDTA used during the passaging process?
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Name: _________________________________________
Culturing and Fluorescent Staining of B16 melanoma cells
Passaging record
Date of passage: _______________
% Confluence: ____________
Name of person(s) doing passaging: _______________________________________
ml of Trypsin/EDTA used: ______________
Incubation time at 37OC: ________________
Cell Counts:
#1
#2
Calculation of cell density:
_____
_____
_____
_____
_____
_____
_____
_____
Calculation of volume containing 104 cells:
_____
_____
Totals: _____
_____
Avg: _____
Replicate this table below for each passaging
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Name: _________________________________________
Culturing and Fluorescent Staining of B16 melanoma cells
Week 3 - Rhodamine-phalloidin staining
Answer questions A - D before coming to lab
A. What are f-actin and g-actin? How are they related?
B. What is the purpose for each of these reagents in the staining process?
Rhodamine:
Phalloidin:
Fixative:
Permealization buffer:
DAPI:
C. What is the fundamental property of a molecule that acts as a fluorochrome?
D. In the fluorescence microscope, what are the functions of the…
Barrier filter:
Excitation filter:
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Name: _________________________________________
Culturing and Fluorescent Staining of B16 melanoma cells
Rhodamine-phalloidin staining, cont.
Record the steps of the procedure as you perform them; the emphasis should be on the
quantities, time periods, volumes
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Name: _________________________________________
Protein Measurement through Absorption Spectrophotometry
Answer questions A - D before coming to lab
A. Why are you preparing a liver homogenate in this lab exercise; how will it be used later in the
upcoming lab exercises?
B. What causes ‘light scattering’ and how does it affect absorbance measurements?
C. Explain how a protein standard curve is created and used to determine the protein
concentration of an unknown. Include in you explanation, the purposes of “BCA” and “BSA”.
D. In protocol #2, Measuring the protein concentration of the homogenate, you will dilute your
liver homogenate. Why? Determine the volumes that you will need to use and right them here and
in the lab manual is the space provided.
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Name: _________________________________________
Protein Measurement through Absorption Spectrophotometry
Procedures
You can cite the procedures from the lab manual, (“Procedure as described in ….”)
but record all the quantitative values (weight of liver, length/speed of centrifugation, volumes
of solutions) and record any deviations from the cited procedure. – be thorough!
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Name: _________________________________________
Protein Measurement through Absorption Spectrophotometry
Results
♦♦♦ Print on or tape to the back of pages your final graphs ♦♦♦
Complete the following tables to record your results
Table 1. Data for BSA protein assay
Tube #
μg/μL BSA
Abs @ 562 nm
1
Slope of trendline = __________ :L cm-1 :g-1
Table2. Data for Liver homogenate replicas
μg/μL protein
Replica #
Abs @ 562 nm
(from standard curve)
(blank)
Tara to 0
0
1
Avg
------
________ μg/μL
Protein concentration in the original homogenate = _________
Volume of homogenate containing 50 μg protein:
______ μL
**Copy this value to page 4 (A.Sample preparation, step 3B) of the Electrophoresis and
Electrooblotting lab exercise**
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Name: _________________________________________
Protein Measurement through Absorption Spectrophotometry
Results, cont.
Table 3. Absorption spectrum measurements (20 nM increments)
Absorbance
Absorbance
Wavelength
BCA only
BCA + BSA
420 nm
8max lies between ________ and _________ nM
Table 4. Absorption spectrum measurements (5nM increments)
Absorbance
Absorbance
Wavelength
BCA only
BCA + BSA
Observed 8max for BCA reagent + protein: ___________
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Name: _________________________________________
Gel electrophoresis and electroblotting
Answer questions A - F before coming to lab
A. Purpose of this and next week’s exercises:
B. Identify the three protocols in this and next weeks’ labs and their purposes:
1.
2.
3.
C. What is the concentration of protein in your liver homogenate:
D. What is the volume of homogenate containing 50ug of protein (show calculations):
E. Give the volumes of each component that will be combined to prepare your liver homogenate
for electrophoresis:
A. ____ μL SDB
B. ____ μL liver homogenate
C. ____ μL VAB
F. What are the functions of these components of the SDB:
SDS:
Brome-phenol-blue:
Β-mercaptoethanol:
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Name: _________________________________________
Gel electrophoresis and electroblotting, cont.
Create a table with each lane number, the samples that were loaded and how they will be later
treated: (although these specific lane number should not be included in the lab report).
Lane
sample
eventual treatment
How long was the gel run for, at what current:
For how long was electroblotting performed, and at what current
Describe the appearance of your Amido black stained gel
Create here the table for data from the MW Standard Curve (see Table 1)
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Name: _________________________________________
Review questions: Type responses
1. Why do the peptides move through the gel only according to their molecular weight rather
than due to charge, shape and subunit structure?
2. What factors (other than the properties of the proteins themselves) would effect the rate of
movement of the proteins through the gel.
3. What is a dalton and approximating how many AAs are present in a protein of 50 Kd? Show
how this was calculated.
4. During the electroblotting procedure what would be the result if the orientation of the
polyacrylamide gel and the nitrocellulose membrane were inadvertently reversed relative to
the electrodes?
5. Why must the proteins be electroblotted to nitrocellulose before probing with antibodies;
i.e., why do we not probe the gel directly?
6. Why is it necessary to ‘block’ the nitrocellulose membrane before performing the Western
blotting procedure? What would happen during the immunoblotting procedure if this step
were left out?
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Name: _________________________________________
Immunoblotting
Answer A – D before coming to lab
A. Blocking buffer: purpose:
B. Primary Ab: purpose, type, concentration, and incubation time:
C. Secondary Ab conjugate: purpose, type, concentration, and incubation time:
D. Staining reagent: type, explain why it changes color:
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Name: _________________________________________
Immunoblotting, cont.
Identify the treatment used for your membrane:
Describe the appearance of your membrane (or if yours was a negative control, than that of another
group with bands):
In the space below identify each control run in this lab exercise, the alternative explanation it is
designed, and which bands appeared
1.
2.
etc
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Name: _________________________________________
Immunoblotting, cont.
Copy Table 1 for immunoblotting results below.
What are the specific conclusions drawn about the results during the class discussion
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Name: _________________________________________
Review questions: Type responses
1. How is a primary antibody, such as “rabbit anti-human β-actin” prepared?
2. What is “goat-anti-rabbit IgG – alkaline phosphatase”?
3. What is the advantage of using secondary antibodies, rather than simply attaching a
detection enzyme such as alkaline phosphatase directly to the primary antibody?
4. What was the conclusion drawn for the experiment relevant to the hypothesis.
Write a numbered list of the evidence that supported this interpretation.
5. Some of the bands in Western blots were not actin. What caused these bands to appear?
6. We identified one of the aberrant proteins; what was it? Write a numbered list of the
evidence that supported this interpretation.
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