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BIOL 2362
FURTHER METABOLISM AND GENE EXPRESSION
LABORATORY MANUAL
Academic Year (2011-2012)
TABLE OF CONTENTS
Page
LAB PREPARATION & REPORT SUBMISSION
CITATION OF REFERENCES
1. PRACTICAL # 1a
Isolation, Quantification and Purity Determination of DNA
from Rat Liver and Kidney and Cows Blood
2. PRACTICAL # 1b
Determination of the Total DNA and RNA Content
in Rat Liver, Kidney and Brain
13
3. PRACTICAL # 2
Induction and Estimation of -Galactosidase Synthesis
In Escherichia coli
21
34
LAB PARTNER:
ID#
Title Of Lab This is given on the lab handout (e.g. Titration Curves)
Aim:
This states the objective(s) of the lab, the aim of the experiment
e.g. To determine., To investigate.
Theory:
being tested, along with the experimental approach that was used. It
puts the experiment into perspective. YOUR INTRODUCTION
MUST BE REFERENCED. Failure to reference your work will
result in loss of marks.
Procedure:
References:
POST-LAB SUBMISSION
NAME:
LAB PARTNER:
ID#
Results: The results section presents the data and observations that bear upon the
objectives of the experiment. Results are presented in tables (PLEASE DO
NOT INSERT DATA SHEETS FROM THE LAB SESSION!), figures,
graphs, calculations, diagrams, and sometimes very brief, accompanying notes.
The following is a guideline:
Tables Must:
1. Be numbered
2. Have a title (this should fully describe what the table is about e.g. Table 1: Results of
Protein Calibration Curve Construction)
1. Be properly drawn (i.e. comprehensive layout of variables)
2. Incorporate all data (e.g. volume of reagents used, absorbance values, amount of
protein in mg etc.)
Calculations:
1. Only do a sample calculation (e.g. if you are constructing a protein calibration curve,
using increasing volumes of standard protein solution, you are expected to show a
calculation showing how much protein there is in a chosen aliquot of solution. You
are not expected to show the working for each aliquot that you pipette out. This is
redundant as the same method is employed.
Alternatively you may be expected to determine the protein content of a specific mass
of tissue. You may be required to do this for several different types of tissues,
however, provided that the dilution factors and processing steps are the same,
you are only required to show the working for one tissue type. Of course even if you
do not show the working, you will include all calculated values in a summary table.
4
2. You must show all steps in your calculations. Marks will be lost if you do not show a
logical flow of steps.
Graphs Must:
1. Be numbered
2. Have a title (this should be fully descriptive e.g. Graph #1: Protein calibration curve
showing how protein content (mg) varies with absorbance at 750 nm)
3. Be neatly drawn (if hand drawn use a sharp pencil point, avoid leaving stray marks
etc.)
4. Possess a scale (e.g. on horizontal axis 1 cm represents 1 unit/mg protein etc.)
3. Have labeled axes (e.g. vertical axis Absorbance at 750 nm; horizontal axis
Protein content in mg)
Discussion:
The discussion is a very important part of the lab script. It demonstrates a
students analytical skills and gives insight into how well the student understands
the lab exercise. This is where you interpret your results and compare it to
findings previously reported in literature.
The discussion should:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Additional Discussion:
Answer these questions separately from the discussion.
*References:
See pgs 5 & 6 of this manual. FOLLOW INSTRUCTIONS RIGOROUSLY!
FAILURE TO COMPLY WITH THE ABOVE INSTRUCTIONS WILL RESULT
IN LOSS OF MUCH NEEDED MARKS!
LAB GRADES
Pre-labs are to be submitted on the day of the lab in question and post labs one week after
the lab date. Each part is individually marked. The mark for a given lab is produced by
summing the totals of the pre and post labs and scaling it down to 10. The marks for all
your lab sessions are then summed and this is added to the scaled-down average of your
in-lab assessments. This final mark is then scaled to 20%, which is your final lab mark
(unless otherwise stated).
Once a week after the submission date has passed, labs will not be accepted unless a very
reasonable excuse is offered.
Your lab grade accounts for 20% of your total course grade. There MAY be a practical
exam at the end of the semester. Otherwise, assessment is based on lab reports,
performance in the lab (e.g. lab manipulation skills, lab conduct, adherence to safety
regulations etc.) and any additional lab exercises (e.g. pop quizzes). Satisfactory
performance in the practicals is a prerequisite for a passing grade. Candidates who do not
obtain an overall passing grade (8 out of the 20%) for the practicals will be required to
repeat the entire course.
COURSE ASSESSMENT
The grade awarded for biochemistry is calculated as follows:
Final examination
In-course Assessment
60%
40%
The in-course assessment comprises of two (2) written term examinations at 10% each
and performance in the practicals worth 20%.
Layout
Fully labeled
tables, diagrams
and figures
Inappropriately
labeled Tables,
Diagrams and
Figures
Exceeds word
limit
Script is difficult
to read
Tables,
Diagrams and
Figures lack
titles
Exceeds or is
severely under
word limit
Illegibly written
Objectives
Materials &
Methods
Introduction
Concise
Based on objectives
All essential
background
ALL goals
information
Past Tense
clearly
Contains necessary
stated
diagrams/
No tables
biochemical
reactions
References cited in
text
Has some main
points
Some
Missing some
objectives
equations or
missing
diagrams
Few in text
references
Past Tense
Has tables
Verbose
Poorly
Lacks important
objectives
reactions
No in-text references
Results
Discussion
References
Concise
All results/
Observations are
analyses are
well explained &
clearly shown &
reinforced by
properly tabulated expected or
Includes all
Correct & easy to published results
cited text
follow sample
Includes
Written as
calculations
suggestions for
indicated in
Clearly
improvement and
manual
documented
important
observations/
precautions noted
diagrams
In-text references
cited
Incoherent layout
of results
Results are
Reference
cited in text
Difficult to follow & partially explained
missing some
but not
sample
included in
Inferences are not
calculations
supported by cited
reference list
references
or vice versa
Incomplete
observations/
diagrams
Results are
missing
In point form
Incorrect & difficult
multiple
to follow sample
tenses
calculations
Includes
Poorly described
tables
observations/
diagrams
Verbose
Trends are
No references
cited in text
Not written as
stated in
manual
no reference
list
Grade
GoodExcelle
nt
Fair
Weak
BIOL 2362
BIOL 2362
Parsons, T.R. 1980. Zooplankton production. In: Barnes, R.S.K. and Mann, K.H. eds.
Fundamentals of aquatic ecosystems. Blackwell, Oxford.
3. Reference to a journal (periodical) article:
Author(s) of article. Year of publication. Title of article. Title of periodical in italics,
volume number (issue number): first page - last page.
Example:
Tedder, T. and Isaacs, C. 1989. Isolation of cDNA's encoding the CD19 antigen of human
and mouse B lymphocytes. J. Immunol. 143(3): 712-717.
4. Reference to an internet source:
Author's name. Title of document, in quotation marks. Title of complete work (if
relevant), in italics or underlined. Date of publication or last revision. URL, in angle
brackets. Date of access in parentheses.
Citing a personal site
Joseph Pellegrino, "Homepage," 12 May 1999, <http://www.english.eku.edu/
pellegrino/default.htm> (12 June 1999).
Citing a professional site
Gail Mortimer, The William Faulkner Society Home Page, 16 September 1999,
<http://www.utep.edu/mortimer/faulkner/main faulkner.htm> (19 November
1997).
National Association of Investors Corporation, NAIC Online, 20 September 1999,
<http://www.better-investing.org> (1 October 1999).
BIOL 2362
Date:
Total
Punctuality
Late (-5)
Safety
Was not wearing gloves (-2)
Improperly worn lab coat (-2)
Inadequate foot ware (-2)
Pours toxic waste down the sink
(-5)
Pours non-toxic waste into waste bottle (-3)
Pools all wastes together, toxic and harmless and then disposes (-3)
Instrument Usage
Holds pipette incorrectly (-3)
Often slams spec cover (-3)
pH meter electrode not replaced in pH buffer (-4)
pH of solution taken without stirring or agitating (-4)
Efficiency
No Flowchart (-10)
Makes one or few careless mistakes (-3)
Makes careless mistakes which renders entire experiment useless (-5)
Has no plan, concentrations/volumes of solutions worked out in advance (-5)
Lab Etiquette & Courtesy
Group takes too much reagents (-3)
Water bath near dried out (-2)
Breakage
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Buffer A
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0.32M sucrose
10 mM Tris HCl
5mM MgCl2
0.75% Triton-X
Adjust pH to 7.6
Buffer B
20 mM Tris-HCl
4 mM Na2EDTA
100 mM NaCl
Adjust pH to 7.4
N.B. All solutions should be sterile. Buffer A should be autoclaved prior to addition of
Triton-X-100. Sterile filtering of solutions instead of autoclaving is a better option.
10
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4. Pipette 2mL of each your liver, kidney and blood stock DNA into separately labeled
test tubes and add 1.5mL 20% (v/v) TCA solution to each. Vortex.
5. Heat all mixtures in a boiling water bath for 15 minutes. Leave to cool at room
temperature.
6. Determine the DNA and RNA concentrations of your liver, kidney and blood samples
as shown on pg 15 step 13 of the manual. DO NOT DILUTE THE EXTRACTS
HERE (lab 1 a) FOR THE RNA PROCEDURE.
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This reagent contains concentrated hydrochloric acid. When RNA is heated with acid in
the presence of ferric chloride (FeCl3.6H2O) as a catalyst, the acid cleaves some of the
phosphodiester bonds and hydrolyzes the glycosidic linkages between sugars and purines.
The hot acid also converts the ribose to furfural, which, when in the presence of ferric
ions, reacts with orcinol to produce green-coloured compounds. The orcinol reaction is
not as specific as the diphenylamine reaction as all pentoses, including the deoxyribose of
DNA, will react to some extent.
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For a given DNA and RNA solution of the same concentration, the colour intensity
produced by the orcinol reaction of DNA is one tenth that of the reaction with RNA.
Therefore the calibration curve for orcinol cannot be used to directly determine the
concentration of RNA in the TISSUE EXTRACT, as this contains a mixture of DNA and
RNA. The absorbance due to DNA is easily estimated once the concentration of DNA in
the extract has been determined. This will be 10% of the absorbance of the same
concentration of RNA on the calibration curve. Once the absorbance due to DNA is
obtained, subtract this from the absorbance reading for the extract. Using the remaining
absorbance value, determine the concentration of RNA from the calibration curve for the
orcinol reaction.
Prelab Questions:
1.
What does treatment with cold TCA and hot TCA accomplish?
2.
3.
What does the RNA to DNA ratio reflect about the constituent cells?
REAGENTS
1.
Diphenylamine reagent
Dissolve 1.0g of diphenylamine in 100mL of glacial acetic acid and add 2.75mL
of concentrated H2SO4, reagent quality.
2.
Orcinol reagent
Mix together 0.2g of orcinol, 60mL conc. HCl and 0.2mL 10% (w/v) ferric
chloride solution (must be fresh).
3.
4.
0.1M KCl
5.
5% TCA
6.
10% TCA
7.
20% TCA
14
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CAUTION!
STRONG ACIDS ARE USED IN THIS PROCEDURE:
DO NOT PIPETTE BY MOUTH
USE GLASS CUVETTES
USE GLOVES AT ALL TIMES
DO NOT POUR REAGENTS INTO THE SINK. THEY WILL EXPLODE ON
CONTACT WITH WATER. POUR ALL REAGENTS INTO THE PROVIDED
WASTE BOTTLE (CONTAINING 1M NaOH), IMMERSED IN THE ICE BATH
LOCTED ON THE CENTRE BENCH.
IT IS ADVISED THAT PROTECTIVE GOGGLES SHOULD BE WORN.
IF ACID SPILLS ON YOUR SKIN, WASH IMMEDIATELY WITH SOAP AND
THEN WATER
PROCEDURE:
A.
1.
Pipette 2mL of the stock DNA solution to a test tube and then add 1.5mL of a
20% (v/v) TCA solution.
2.
Heat the mixture in a boiling water bath for 15 minutes. Leave to cool at room
temperature for 2 minutes and then quickly cool the tubes under the tap. This is
your DNA-acid hydrolysate.
3.
(c)
Prepare a blank containing 0.7mL of water, 0.3mL of 20% TCA and 2mL
of diphenylamine reagent.
(d)
15
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(e)
Cool the tubes and measure the absorbance at 600nm after setting the
spectrophotometer at zero with the reagent blank. Plot Absorbance at
600nm against DNA content (mg).
B.
1.
2.
Heat the mixture in a boiling water bath for 15min. This is your RNA-acid
hydrolysate.
3.
(c)
Prepare a blank to contain 0.7mL of 5% TCA and 0.3mL water and 2mL
of orcinol reagent.
(d)
Mix all tubes thoroughly and then boil for 10 minutes in a waterbath.
(e)
Cool and measure their absorbance at 660nm against the reagent blank.
Plot absorbance at 660nm against RNA content (mg).
C.
Estimation of DNA and RNA in Rat Liver, Kidney and Brain Tissues
1.
Kill one rat (200-250g-body weight) and remove the liver, kidney and brain and
weigh them.
2.
4.
Add 5mL of ice-cold 10% TCA to the centrifuge tube. Mix well by gentle
inversion (place a foil cap over the test tube to protect your finger).
5.
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6.
Using a Pasteur pipette, remove and discard the supernatant. If there is a solid
layer floating on top, save this by inserting the Pasteur pipette beneath the layer
in order to aspirate the supernatant.
7.
Add another 5mL of ice-cold 10% TCA to the pellet and resuspend with the
Pasteur pipette. Centrifuge and aspirate as described in steps 5 and 6 leaving two
drops of liquid above the pellet.
8.
With a Pasteur pipette, disperse the pellet in the liquid above it. Then add 10mL of
95% ethanol (room temperature) to the dispersed pellet.
9.
10.
11.
Add 5.0mL of 5% TCA (room temperature) to the centrifuge tube and disperse the
pellet with a Pasteur pipette. Place the tube in a boiling water bath for 10 minutes
agitating every few minutes.
12.
Centrifuge at 1300g for 5 minutes and carefully decant the supernatant in a clean
test tube marked A (12 mL capacity). MEASURE THE VOLUME OF THE
SUPERNATANT. This nucleic acid extract will be used in the assays.
13.
Determine the DNA and RNA concentrations in the acid hydrolysates as follows:
(a)
(b)
17
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18
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LAB #1a & 1b Tabulate the data for questions 2 and 3. Clearly distinguish between
the samples prepared in Lab 1a and 1b.
1.
Determine the colour contribution of DNA to the orcinol reaction of the extract.
(DNA reacts one-tenth as much as the same concentration of RNA). From the
calibration curve for the orcinol reaction, find the absorbance value for an RNA
concentration that is the same concentration as the concentration of DNA in the
extract. Take one-tenth of that absorbance as the absorbance component due to
DNA and enter the values.
2.
Calculate the DNA and RNA concentration (mg/g tissue) for all your samples.
3.
Determine the total DNA and RNA in each organ for all your samples.
4.
Do the different tissues contain different amounts of DNA and RNA? In which
tissue is the RNA concentration the highest? In which tissue is the DNA
concentration the highest? Explain the significance of your results.
5.
Discuss the advantages and disadvantages of both procedures (Lab 1a and 1b) as
quantitative methods to determine DNA concentrations.
6.
Compare the values acquired for liver and kidney tissues (mg/g) in Lab 1a and
Lab 1b. Explain your differences. Comment on your results.
REFERENCES:
1.
2.
Roger, L.P., Adams, John T Kowler & David P Leader. The Biochemistry of the
Nucleic Acids, 10th Edition.
Harpers Biochemistry
19
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DATA SHEET: LAB # 1 a & b
DETERMINATION OF THE TOTAL DNA & RNA IN RAT LIVER, KIDNEY AND
BRAIN TISSUES
NAME:.LAB PARTNER:
LAB#1a
ABSORBANCE READINGS FOR DNA FROM LIVER, KIDNEY & BLOOD
Tissue
260 nm
600 nm
660 nm
0.5mL 1.0mL 0.5mL 1.0mL
280 nm
Liver
Kidney
Blood
LAB #1b
A.
1
(blank)
mL DNA-acid
--hydrolysate
A600nm
0.00
DNA content
(mg)
B.
0.1
0.2
0.2
0.3
0.5
0.5
0.8
0.00
1
(blank)
mL RNA-acid
--hydrolysate
A660nm
0.00
RNA content
(mg)
0.1
0.2
0.2
0.3
0.4
0.4
0.5
0.00
20
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21
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C.
ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BLOOD (1a)
LIVER
Mass (g)/Volume
(mL) of Tissue
A600nm
(DNA)
A660nm
(RNA)
D.
BRAIN
---0.5 mL
1.0 mL
0.2 mL
0.4 mL
ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BRAIN (1b)
LIVER
Mass of tissue (g)
----
----
A660nm
(RNA)
E.
KIDNEY
BRAIN
KIDNEY
0.5 mL
1.0 mL
0.2 mL
0.4 mL
Lab 1a DNA
Lab 1a
Homogenates Lab 1a RNA
Lab 1b DNA
Lab 1b
Homogenates Lab 1b RNA
22
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(b)
Note: Synthesis of inducible enzymes can often be repressed by the end product of the
metabolic pathway.
Enzyme-catalyzed reactions may be followed in many ways depending on the nature
of the system under study. For example, one can monitor the rate of disappearance of
a reactant or the formation of a product, the rate of gaseous exchange, the effects of
coenzymes etc.
The activity of the enzyme -galactosidase can be readily estimated through
colourimetry. It is an inducible enzyme found in the bacterium, Escherichia coli,
specified by the Z gene. The enzyme catalyses the hydrolysis of the disaccharide,
lactose, releasing -D-glucose and -D-galactose. The assay method involves the use
of an artificial chromogen substrate, o-nitrophenyl--D-galactopyranoside (ONPG),
the hydrolysis of which yields o-nitrophenol (ONP). In alkaline solution, ONP has an
intense yellow colour with an extinction maximum at 420 nm.
Reagents
1. LB Broth (1L)
Composition: 10g Tryptone (or peptone), 5g Yeast Extract, 10g NaCl, pH 7.4.
Sterilize for 20 min in an autoclave.
2. Mineral Salts Medium (1L)
Composition: 8g K2HPO4, 3g KH2PO4, 0.1g MgSO4.7 H2O, 1g (NH4)2SO4, 0.1% Yeast
Extract, 5% glucose (or lactose), pH 7.0. Sterilize glucose (or lactose) separately for 20 min.
EXPERIMENTAL
23
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A.
1.
2.
Using this standard solution set up a series of tubes containing varying amounts of
ONP and a constant amount of alkali as outlined in the table below.
TUBE NO.
1
2
3
4
5
6
7
Na2CO3 (mL)
1.00
1.00
1.00
1.00
1.00
1.00
1.00
3.
Mix the contents of tubes thoroughly, measure absorbance at 420nm using the
contents of tube 7 as a reagent blank.
4.
Q. 1
N.B.
B.
(i)
E. coli cells have been grown in LB Broth (25 mL) overnight. The suspension was
centrifuged and the cells resuspended in 2 mL sterile water. 1 mL was added to glucose
media (50 mL) and 1 mL to lactose media (50 mL). These were then left to grow
overnight (or approximately 18 hours). Note: these quantities quoted are for each pair of
students.
24
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6.
7.
8.
9.
10.
For the tube labeled glucose A, resuspend the pellet in 2 mL of sterile water. For
the tube labeled lactose A, re-suspend the pellet in 6 mL of sterile water.
11.
From each of these tubes, take 1 mL of suspension and place into two new test
tubes labeled glucose B and lactose B respectively.
12.
To the new tubes (labeled B)add 1 mL toluene. Vortex the toluene tubes for 3
min.
(ii)
13.
Pipette 4 mL of 0.1 M phosphate buffer (pH 7.2), into each of four new test tubes
labeled glucose A, lactose A, glucose B and lactose B,
14.
15.
Place the test tubes in a water bath at 37 C and allow the contents to attain this
temperature (wait 5min).
16.
17.
Shaking the tube beforehand, remove 0.5 mL samples at specific time intervals (0,
5, 10, 20, 30, 40, 50, 60 min).
18.
19.
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20.
21.
Carefully transfer the supernatant to the cuvette and read the absorbance at 420
nm. Use 0.5 mL 1 M sodium carbonate and 4.5 mL water as your blank.
26
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Treatment of data
Determine the activity (units/mL) of your four stock samples.
Calculate and compare the units/ml of enzyme in each of your stock samples.
Also plot ONP concentration (umol/ml) against time (mins) and draw tangents at the
linear portions of the curves to determine U/ml (initial velocity) for each sample.
Discuss your results fully.
REFERENCES:
1.
2.
Hestrin, S., Feingold, D. S., & Schramm, M. Methods in Enzymology. Vol. I. Ed.
P. Colowick & N. O. Kaplan. p. 241.
3.
4.
Robert F. Boyd (1988) General Microbiology. 2nd Ed. Times Mirror, Mosby
College Publishing p. 237-238.
27
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NAME:..LAB. PARTNER:..
A.
[ONP] moles/mL
A420nm
*reagent blank
B.
Time (min.)
0
5
10
20
30
40
50
60
Glucose A
Absorbance at 420nm
Glucose B
Lactose A
28
Lactose B
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Absorbance reading at X nm
0.05
0.16
0.225
0.33
0.375
The best thing to do is to change the units to easier numbers to be plotted. For example,
multiply the values by 1000 so that you obtain readable values like 1, 3, 5, 7 and 8. Then
on your graph you can use exponents to reflect this. The x-axis would now be labelled
mg x 1000 or mg x 103. Additionally if your values are very large numerically, one can
simply find the log10 value and plot points using a log scale. Remember to take the
antilog for any value obtained from the graph that will be used in further calculations.
VARIABLE RANGE
In order to determine an appropriate variable range; subtract the lowest data value from
the highest data value. Do each variable separately.
29
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SCALE
Never forget to insert the scale for both axes, as the two may vary. Additionally the scale
must be constant. If you define 1cm on the x-axis to be 1g then that scale must be
maintained.
LABEL AXES
Ensure that both axes are clearly labelled. Axes titles should always include the name of
the variable as well as the unit the variable is measured in e.g. Protein content (g). NB
please insert arrowheads at the end of the axes to indicate the direction of progression.
PLOTTING DATA POINTS
Data points for a single data series can be plotted using any one of the following symbols:
x, +, ,,,,, etc. In order to plot more than one data series on a graph, one must use
different symbols to distinguish between different lines (one can also use lines of
different colours). Insert a key to distinguish between different data series.
30
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Figure 1. (a) scatter plot (b) line of best fit. NB that the best-fit line does not necessarily pass
through any of the points plotted.
Absorbance @ 750 nm
0.5
0.4
0.3
0.1
0
0
13
25
38
31
50
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TITLE:
Your graph must be numbered and have a descriptive title. For example, Graph #1:
Protein Calibration Curve.
B.
There are many graphing programs available but Excel is the most popular choice at this
level. Therefore we will use Excel to demonstrate how you can analyse data using
spreadsheets and graphing programs.
Loading the Spreadsheet
The first step is to set up the spreadsheet. With Excel you have a basic grid of columns,
which are labelled A-Z, and rows, which are numbered one to several hundred. Each
combination of a number and letter is called a cell. You start by putting data into the cells.
Cells can hold data in the form of numbers or letters. If you wanted to plot absorbance vs.
mg of protein for the Lowry assay, you might have data that looks like Table 2.
When you load this into Excel, you could put the column labels (mg Protein and
Absorbance into the spreadsheet. The program will talk you through the creation of the
graph, including labelling the axes. It is best to put the values that will be on the x-axis
into the left-hand column, as it simplifies the graphing process. Figure 3 shows what the
spreadsheet would look like.
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30
40
50
0.29
0.40
0.52
33
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determines the number of visible gridlines. The fourth dialog box gives you the option of
putting the graph as an object on the spreadsheet itself or making it a separate page.
SELECT THE LATTER.
Remember to always select for a graph that goes through the origin when plotting
calibration curves.
To avoid creating the default connect the dots graph, we selected a graph that had no
line. You therefore still need to connect the line. Create the line using the pulldown
menus on the top bar. Choose chart and then add trendline choice from the pulldown
menu. The box that pops up gives you a picture of possible types of lines, including linear
regression and polynomial. Select linear regression for linear data.
Non-Linear graphs
Such graphs would start off the same way but you would use the chart wizard differently
to customise your graph. A common example would be when analysing enzyme kinetics
data.
35
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DATA SUBMISSION SHEET: LAB # 1a & b
DETERMINATION OF THE TOTAL DNA & RNA IN RAT LIVER, KIDNEY AND
BRAIN TISSUES
NAME:.LAB PARTNER:
LAB#1a
ABSORBANCE READINGS FOR DNA FROM LIVER, KIDNEY & BLOOD
Tissue
Mass (g)/
Volume (mL)
260 nm
600 nm
660 nm
0.5mL 1.0mL 0.5mL 1.0mL
280 nm
Liver
Kidney
Blood
LAB #1b
F.
1
(blank)
mL DNA-acid
--hydrolysate
A600nm
0.00
DNA content
(mg)
G.
0.1
0.2
0.2
0.3
0.5
0.5
0.8
0.00
1
(blank)
mL RNA-acid
--hydrolysate
0.1
0.2
0.2
0.3
0.4
0.4
0.5
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A660nm
0.00
RNA content
(mg)
0.00
37
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H.
ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BRAIN TISSUES
LIVER
Mass of tissue (g)
----
----
A660nm
(RNA)
I.
BRAIN
KIDNEY
0.5 mL
1.0 mL
0.2 mL
0.4 mL
ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BRAIN (1b)
LIVER
Mass of tissue (g)
----
----
A660nm
(RNA)
J.
BRAIN
KIDNEY
0.5 mL
1.0 mL
0.2 mL
0.4 mL
Lab 1a DNA
Lab 1a
Homogenates Lab 1a RNA
Lab 1b DNA
Lab 1b
Homogenates Lab 1b RNA
38
BIOL 2362
NAME:..LAB. PARTNER:..
C.
[ONP] moles/mL
A420nm
*reagent blank
D.
Time (min.)
0
5
10
20
30
40
50
60
Glucose A
Absorbance at 420nm
Glucose B
Lactose A
39
Lactose B
BIOL 2362
40