Lab Manual General Guidelines

BIOL 2360 Biochemistry II
BIOL 2360
Lab manual
General guidelines
A. INTRODUCTION
Biochemistry is the chemistry of biological systems. The practical component of
biochemistry is aimed at developing your interest in and understanding modern
biochemical and molecular biological experimentation. The techniques learnt in the
biochemistry lab will be applicable to all life sciences.
THE OBJECTIVES OF THE BIOCHEMISTRY LABORATORY INCLUDE:
(1) Learning the theory behind the techniques and biochemical pathways
(2) Learning the physical skills and techniques of modern experimental biochemistry
(3) Learning how to THINK SCIENTIFICALLY and INDEPENDENTLY and
mastering routine calculations.
You are assisted in meeting these objectives by the following:

a. Each experiment is preceded by a short introduction designed to familiarize you
with the theory and techniques underlying the lab.
b. The experiment is followed by a set of additional exercises and references that
allow you to develop further understanding of the particular method, technique
and theory of the lab.
c. The laboratory session often follows a lecture that covers the principle of the lab.
The experiments have all been tested thoroughly and tried by hundreds of students who
have been successful. The experiments have been designed so that they can be completed
within the allocated time with enough time to wash all glassware, submit data sheets
and clear the bench tops.
Your success in these lab sessions will depend on your ability to understand the
experimental procedures and organize yourself so that you make efficient use of
your time. IT IS THEREFORE IMPERATIVE THAT YOU READ AND
FAMILIARIZE YOURSELF WITH THE PROTOCOL BEFORE YOU ENTER
THE LAB.
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SAFETY GUIDELINES
The laboratory can be a potentially hazardous place if you are not careful. Some of the
chemicals used are very dangerous and care must be taken when handling them. Broken
glassware (due to student carelessness) is a potential hazard as flying pieces can come
from anywhere. Proper clothing and eye protection is the sensible way to protect yourself
against most common lab accidents. A student who is mentally prepared to undertake the
lab, has studied the material and understands the procedures is less likely to make a
mistake or injure someone.
THINGS TO DO
A. Always wear a long sleeved, calf-length lab coat in the lab. You will not be
permitted into the lab without a lab coat.
B. Wear proper clothing in the lab. That means, wear long pants/skirts and close toed
shoes. The use of protective eyewear is also advised. You will not be permitted
into the lab if sandals are worn or your toes are exposed.
C. Be aware of the chemicals you will be using. This means, consulting texts, which
list toxicity and safety, concerns. Two such texts are “Prudent Practices in the
Laboratory” and “The Merck Index”. Additionally the Material Safety Data
Sheets (MSDS) for the chemicals that you will be using can be obtained online
using a Google search (www.google.com).
D. Familiarize yourself with the layout of the lab. Know where the exits are, the fire
extinguishers, the first aid kit, eye wash stations etc.
E. Maintain a complete research lab notebook containing all data, calculations, tables
and results for each lab session. This book will be reviewed randomly throughout
the semester.
THINGS YOU SHOULD NEVER DO

1. Never eat, drink or smoke in the lab. Many types of experiments are run in the
lab. Consider that fumes may be lingering in the lab. If you are discovered
eating, drinking or smoking in the lab you will be evicted immediately.
2. Mouth pipetting is prohibited. Mechanical pipettes are provided.
3. Never work alone. You must always have someone else, i.e., a lab partner
working with you.
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LAB COURTESY
In the Biochemistry lab we like to practice good lab etiquette. The following items WILL
make your labs run more smoothly and lead to efficient transitions between lab sections:
1. NEVER stick your personal pipettes into a community reagent bottle. If your pipette
is dirty, you will have just contaminated the supply for the whole class. If you need
10mL of a reagent and there is a 1L bottle in a community reagent area, take a small,
labeled beaker from your cupboard over to the community bottle and pour
approximately 10mL into your personal beaker for your use only. That way if your
beaker is dirty you will have contaminated your own supply and very importantly you
will not have wasted reagent.
2. NEVER take a community reagent back to your own bench. It will become
frustrating when you cannot find something you need.
3. DO NOT USE MORE CHEMICALS THAN YOU NEED. Chemicals are

prepared in advance for use by the entire class. It is incredibly frustrating to approach
the end of the lab and be held up by lack of reagents. Read your lab scripts and
determine precisely the volumes of reagents required. Make small notes to
yourself.
4. ALWAYS clean up your lab area and any equipment and glassware you used. The
next class may need to use the same materials. You will be penalized, i.e. marks
will be deducted from your lab if you fail to sufficiently tidy your workplace.
MISSED LABS
Lab attendance is taken at each lab session. You must have a valid reason for missing the lab
and written proof is required. In the case of illness an original medical must be submitted to
Administration, a copy to the Dean’s office and a copy to the Biochemistry office. The
Teaching assistant must be notified. Outside of valid medicals, all reasons will be assessed on
an individual basis. There is NO makeup of missed lab sessions. BEING ABSENT
FROM A LAB SESSION DOES NOT EXEMPT YOU FROM SUBMITTING A LAB
REPORT.
LAB PARTNERS
You will work only in pairs in the lab with the exception of an odd number of students. The
T.A. will assign you into pairs IF this has not been established prior to the lab session. Both
lab partners must record data from the lab. Both lab partners must submit an independently
written lab report.
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LAB PREPARATION & REPORT SUBMISSION

FLOWCHART
As part of your preparation for the lab session, you are required to come to the lab with a
comprehensive flowchart outlining the procedures and your plan to undertake the lab
session in question. On the top of the page of the flowchart you should have:
 The volume of each reagent you are required to take.
 Major notes/reminders
 Expected trends/results for a given experiment.
 Questions (if any) about the procedure
Absence of your flowchart will result in an immediate deduction of marks.
IN-LAB ASSESSMENT
Your lab skills and ability to produce accurate results are assessed in the in-lab
assessment. Every lab session, both you and your partner will be assessed. The final mark
will be acquired by summing the totals of all the assessment and scaling it down to an
appropriate mark which is then included in your final lab mark.
LAB REPORT SUBMISSION
BIOCHEMISTRY LAB REPORTS SHOULD BE TYPED (PREFERABLY) ON

LETTER SIZE PAPER OR WRITTEN LEGIBLY ON FOLDER/BINDER PAGES.
FOLLOW THE GUIDELINES BELOW STRICTLY.
NAME: LAB PARTNER:
ID# DATE (of lab session):
Course Code
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: The theory should pertain to the objectives of the lab. It is a

concise introduction that gives information needed to understand
the subject and objectives of the experiment. YOUR
INTRODUCTION MUST BE REFERENCED. Word limit,
unless otherwise stated, is 400 words. Your recommended text is
usually a good start to locate information for your introduction.
Failure to reference your work and stay within the word limit will
result in loss of marks.
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Procedure: The procedure must be presented in narrative style in the PAST

TENSE. DO NOT ITEMISE OR PUT IN PUT IN POINT FORM.
THERE SHOULD BE NO TABLES IN THE PROCEDURE.
Include a list of materials and reagents used at the beginning of this
section.
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)
3. Be properly drawn (i.e. comprehensive layout of variables)
4. 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.
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.)
5. Have labeled axes (e.g. vertical axis – Absorbance at 750 nm; horizontal axis –
Protein content in mg)
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Discussion:
The discussion is a very important part of the lab script. It demonstrates a student’s
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. Answer the questions raised in your Aims/Objectives.

2. FOCUS ON YOUR RESULTS!!!!!
3. Show significant trends in the results
4. Point out the significance of the results/trends by comparing your findings to known
theoretical values or theories.
5. State and discuss whether the results obtained conform to the theoretical expectations
and if they do not suggest possible explanations.
6. Account for sources of error and indicate why certain precautions may have been
taken. (e.g. tissue samples kept on ice)
7. Indicate why certain reagents were used (if the addition of such reagents are
important for the particular reaction).
8. BE REFERENCED to support your findings.
9. Give suggestions for improvements in experiments.
Additional Discussion:
Answer these questions separately from the discussion.
*References:
See pgs 8 & 9 of this manual. FOLLOW INSTRUCTIONS RIGOROUSLY!
FAILURE TO COMPLY WITH THE ABOVE INSTRUCTIONS WILL RESULT

IN LOSS OF MUCH NEEDED MARKS!
LAB SUBMISSION
Lab reports are due exactly one (1) week after the day of the lab. Reports are to be submitted
between 8.00 AM and 3:30 PM to the secretary in Room 214. You must sign the sheet attached to
the envelope while the secretary confirms your submission by placing a dated stamp on your
report.
MARKS WILL BE DEDUCTED (0.5 MARK PER DAY) FOR LATE SUBMISSION. Once
a week after the submission date has passed, labs will not be accepted unless a very reasonable
excuse is offered. Lab marks will be posted on myelearning so students could confirm that the
marks entered are correct.
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Students are required to submit their introduction and discussion to the Turnitin link via BIOL
2360 myelearning group. A similarity report of <10% is expected. >10% will be considered
plagiarism.
NB Plagiarism is treated very harshly (whether the source of materials

is the Internet or a colleague’s report). If you are discovered plagiarizing you will
receive a mark deduction based on the level of plagarism. High levels of plagiarism
will result in no marks awarded.
LAB GRADES
Your lab grade accounts for 10% of your total course grade. 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).
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General Guidelines For A Good Lab Report
Materials &
Layout Objectives Introduction Results Discussion References Grade
Methods
 Concise
 All results/analyses are
 Concise  Observations are well
 Fully labeled tables, clearly shown & properly
diagrams and figures
 ALL goals
clearly stated
 Based on objectives
 All essential background
information
 Past Tense
tabulated
 Correct & easy to follow
explained & reinforced by
expected or published
results
 Includes all cited text
 Written as indicated
Good-
 Within word limit sample calculations
 Legible, justified report

 Contains necessary diagrams/
biochemical reactions
 References cited in text
 No tables
 Clearly documented
observations/diagrams
 Includes suggestions for
improvement and important
precautions noted
in manual
Excellent
 In-text references cited
 Results are partially
 Inappropriately labeled  Reference cited in
 Incoherent layout of results explained
Tables, Diagrams and  Has some main points text but not
 Past Tense  Difficult to follow & missing


Figures
Exceeds word limit
Script is difficult to
 Some objectives
missing
 Missing some equations or
diagrams
 Few in text references
 Has tables
some sample calculations
 Incomplete
 Inferences are not
supported by cited
included in
reference list or
vice versa
Fair
observations/diagrams references
read
 Tables, Diagrams and  Verbose
 Verbose
Figures lack titles  Results are missing  Trends are reported but
 Poorly  Lacks important background  Not written as
 In point form  Incorrect & difficult to not explained
 Exceeds or is severely
under word limit
constructed and
missing
objectives
points
 Plagiarism detected
 No diagrams/reactions
 multiple tenses
 Includes tables
follow sample calculations
 Poorly described
 Too much focus on
precautions

stated in manual
no reference list
Weak
observations/diagrams  No references cited in text
 No in-text references
 Illegibly written
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B. CITATION OF REFERENCES FOR THE BIOLOGICAL SCIENCES

Because all the sources of information provided in your biochemistry labs (introduction
and discussion) were not your original work, it must ALL be
referenced. The Chicago Manual of Style describes methods that are frequently
used by the Biological Sciences. We in Biochemistry will accept the author-date system
of referencing.
AUTHOR –DATE SYSTEM
MAKING A CITATION WITHIN THE TEXT:

 At every point in the text at which reference is made to a particular
document, include the author's surname and the year of publication in
parentheses.
e.g. In a recent study (Smith 1982), it was shown that .....or alternatively
the reference can be found at the end of the sentence e.g. …..as previously
described (Smith 1982).
 If the author's name occurs in the sentence, give the year of publication
alone.
e.g. Smith (1982), discusses the .....
 If there are two or three authors, give the surnames of all individuals. If
there are more than three, give the surname of the first followed by et al.
e.g. Smith, Lopez and Martin (1982) reported that....
e.g. In an earlier study (Smith et al. 1982) .....
CITING REFERENCES IN A BIBLIOGRAPHY:

 Bibliography should be arranged in alphabetical order by author's surname
followed by year of publication.
 Use the same punctuation.
The following lists examples of citations from different sources.
1. Reference to a book:
Author(s)/editor(s). Year of publication. Title of publication in italics. Edition (if not 1st).
Publisher, place of publication. (page no. optional)
Example:
Fletcher, R. and Voke, J. 1985. Defective colour vision. Hilger, Bristol.
2. Reference to a chapter in a book:

Author(s) of chapter. Year of publication. Title of chapter in italics. In:
Author(s)/editor(s) of book. Title of book. Edition (if not 1st). Publisher, place of
publication.
Example:
Parsons, T.R. 1980. Zooplankton production. In: Barnes, R.S.K. and Mann, K.H. eds.
Fundamentals of aquatic ecosystems. Blackwell, Oxford.
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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).
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LAB SKILLS ASSESSMENT SHEET

Names: Date:
Cupboard #
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) Leaves used tip on pipette (-2)
Often slams spec cover (-3) Spills sample in spec (-4)
pH meter electrode not replaced in pH buffer (-4) pH meter electrode placed on desk (-4)
pH of solution taken without stirring or agitating (-4)
Efficiency
No Flowchart (-10)
The last or in the last 3 groups to leave (-2)
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 more than half filled (-2)
Water bath near dried out (-2) Flasks and Tubes not labeled (-2)
Accuracy & Understanding

Student does not understand what he/she is doing (-3)
Some results are off (-1)
All results off (-3)
Cleanliness
Tips on desk (-3) Miscellaneous paper, parafilm on desk (-2)
Pipettes on bench when not in use (-1) Left dirty utensils on desk at end of lab (-3)
Places dirty utensils in cupboard (-5) Leaves workspace dirty/wet (-3)
Leaves equipment on/plugged in (-3)
Breakage
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C. QUANTITATIVE ANALYSIS
Scientific Notation
Scientific notation is used in biochemistry in order to accurately and clearly communicate
numerical information. Any number in strict scientific notation begins with one non-zero
digit followed by a decimal point and some other numbers as well as an exponent that
tells to what power to raise it.
For example, the number 623 would be written 6.23 X 102. The number 0.0678 would be
written as 6.78 X 10-2.
Avoid starting a number with a decimal point. Thus rather than writing .789, it is
recommended to write 0.789 or 7.89 X 10-1.
Statistics and scientific measurements

For every determination in the laboratory there is a degree of uncertainty in the result. For
example, if a blood glucose result is 100mg/mL, it is obvious that it is not 100.00 mg/mL.
Furthermore if the determination was repeated by the same analyst or by another analyst,
the result may deviate from that of the first analysis. All measured quantities are
variables; it is therefore essential that the analyst should have some conception of the
subject of variability and the many contributing factors. Statistical analysis is used to
quantitate this variability and the methods are designed to answer, among other things,
the following:
(i) The extent of the variation about the average.

(ii) Is a single determination (e.g. blood glucose) far enough from the average value
to indicate abnormality?
(iii) Is there a significant difference in the results obtained by methods A and B and
are the values in one set of data significantly higher than the values in the other
set (e.g. normal range of blood glucose for males vs. females)?
It should be noted that in biochemical analyses variability in results usually originates

from two sources viz.: - biological and experimental.
Population Distributions
The simplest way to describe a population of data is to construct a histogram, also called
a frequency diagram or frequency distribution. Using blood glucose as an example, the
horizontal axis is glucose concentration divided into convenient ranges and the vertical
axis is the frequency (or relative frequency) with which results in each range are
obtained. The larger the number of observations (N), the more representative of the
population the histogram becomes.
There are two basic types of statistical measures. The first is a measure of the central
tendency. The one most often seen is the average or arithmetic mean (Xavg). The
average is calculated by adding up all of the numbers and dividing by the total numbers
you had.
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Arithmetic mean (Xavg) = X1 + X2 + X3 + ----Xn = ∑Xi

n n
A second type of statistical measurement is a measure of dispersion. These usually

measure the variations in the values compared to the mean. Often such a measure is
necessary to truly understand the data represented.
Consider the following example:

If a professor of a class taking an exam notices that of his 200 students, 180 of them
scored an 85 on the exam, with a few being higher and a few being lower, so that the
average was still 85, then he will know that he has a very homogeneous class. Everybody
did about the same with just a few exceptions. On the other hand, if the scores were
scattered all over, with lots of students scoring below 40 and lots above 90, then he will
know that the class is heterogeneous. The professor will then adopt different teaching
strategies to each class. When grades are assigned on a curve, it is always the mean and
some measure of dispersion that determines the grade.
There are many different measurements of dispersion, and the exact reason for using one
or another is beyond the scope of this manual. Some of the more common ones are
presented next:
Mean deviation = ∑ |xi – xavg|

n
where n is the number of samples measure and | | means the absolute value of the
difference between the individual value and the mean.
Percent deviation = Mean deviation x 100

Mean
Variance = σ2 = ∑ (xi – xavg)2

n- 1
Standard deviation = σ = √σ2
The mean and the standard deviation are the two most common statistical parameters.
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D. UNITS
The international system of measurements is known as the SI and is based on the MKS
(meter-kilogram-second) system. The base units for SI are given in Table 1.1.
Many other units are derived from SI units, such as the joule (m2 kg/s2), the unit of
energy. Some non-SI units are also frequently used, such as degrees Celsius, pressure in
atmospheres, and so on. The common unit of volume, liters, is not an SI unit because
volume would be cubic meters. A liter is actually a cubic decimeter.
Multiples of the SI units are frequently used in biochemistry and it is important to know
them. Table 1.2 gives the most common multiples. Very few numbers in biochemistry
exist without units. Imagine if you calculate the volume of a something to be 12.34 cm 3,
your answer will be wrong if you just report the number. It will be just as wrong if you
got the number wrong.
Table 1.1 Basic SI Units

Length meter m
Mass kilogram kg
Time second s
Temperature kelvin K
Electric current ampere A
Amount mole mol
Radioactivity Becquerel Bq
Table 1.2 Prefixes for Multiple Units

1012 tera T
109 giga G
106 mega M
103 kilo k
10-1 deci d
10-2 centi e
10-3 milli m
10-6 micro μ
10-9 nano n
10-12 pico p
10-15 femto f
10-18 atto a
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E. CONCENTRATION OF SOLUTIONS
Every experiment that you do will use at least one solution of a chemical. When
chemicals are used as liquids, they can be either pure liquids or solutions. Absolute
ethanol is an example of a pure liquid because every molecule in that liquid is ethanol
with nothing mixed in.
A solution contains a chemical dissolved in another liquid. In this case you must know
what it is dissolved in and how much of it is dissolved. The chemical in the smaller
quantity is called the solute. The liquid it is dissolved in is called the solvent. When you
calculate the amount of solute that is dissolved in the solvent, you have determined the
concentration.
The concentration is very important to the biological or chemical function of the liquid.
A concentration is always the ratio of an amount of a chemical divided by the total
volume. It is important to be able to distinguish values that are concentrations from
values that are not, which are called amounts. Remember that amounts are additives but
concentrations are not. For example if you put 1g of salt in a beaker, that is an amount.
However, if you add 1 L of water, you now have a 1g/L solution, which is a
concentration. If you add 1mmol (millimole) of salt to a beaker, that is an amount BUT if
you add 1L of water to it, you now have a 1mM (millimolar) solution, which is a
concentration.
% Solutions
Solutions based on percent are the easiest to calculate because they do not depend on a
knowledge of the molecular weight. Your instructor can give you a tube with an
unknown white powder and tell you to prepare a 1% (w/v) solution and you can do it
without knowing anything about the white powder.
% (w/v) means percent weight to volume and has units of grams/100mL. Therefore a 1
% (w/v) solution has 1g of solute in a total of 100mL of solution.
% (v/v) means percent volume to volume and has units of mL/100mL. Therefore a 1 %
(v/v) solution of ethanol has 1mL of pure ethanol in 100mL of total solution.
You can assume, unless told otherwise, the solvent is water for any solution.
Remember that when you calculate concentrations, the important thing is the final
volume of the solution, not necessarily the amount you add. If you have 100g of a solid
chemical and add 1L of water, the final volume will be more than 1L. All
calculations based on concentration assume you have correctly calculated the final
volume after mixing the solute with the solvent.
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Molar Solutions
The most common types of solutions are molar solutions. A 1M solution means 1 mol of
solute in a total volume of 1L. A 1mM solution has 1 mmol (10-3 mole) in a total volume
of 1 L. A 1 μM solution has 1 μmol (10-6 mole) of solute in a total volume of 1L.
Remember that moles are calculated by dividing grams by the formula weight in grams
per mole. For example, if the formula weight of compound X is 39g/mol, this means that
one mole of compound X weighs 39g. If we have 13g of it we calculate moles thus:
13  39g/mol = 0.33 mol
If we then take the 13g and bring it to 0.5L with water, our molar concentration will be
0.333mol  0.5L = 0.666 mol/L = 0.67 M
Remember the derivatives of molar solution, such as mM, M, nM and so on still refer to
the amount divided by litres of solution. Many students mistakenly think that a 1mM
solution means 1mmol in 1mL. This is incorrect. It actually means 1 mmol in 1L!
Dilutions
When starting with a dilution, you always start with a concentration of something and
add more solvent to it, thereby lowering the concentration. There are two simple ways of
doing all dilution problems. The most popular method with non-mathematicians is:
C1V1 = C2V2
Practice: You have a sodium phosphate buffer at concentration of 0.2M. You take
10mL of the buffer and add it to 90mL of water. What is the final concentration after
dilution?
To use the formula, you need 3 of 4 variables.
C1 = initial concentration, which in this case is 0.2M

V1 = initial volume which is 10mL
C2 = final concentration, which is what we want to know
V2 = final volume. The final volume is 100mL.
C1V1 = C2V2
(0.2M)(10mL) = C2(100mL)
C2 = (0.2M)(10mL)/100mL = 0.02M
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Dilution Factors
The second way is faster and is better for multiple dilutions. The dilution factor is the
final volume divided by the initial volume. Therefore, in the previous example, the
dilution factor is 100mL/10mL = 10, or a 10 to 1 dilution, or a 1 to 10 dilution.
F. GRAPHING
DRAWING GRAPHS BY HAND

A graph is a plot of two quantities. One is something that you control and is called the
independent variable. Usually it goes on the x-axis. The other quantity that changes as
you change the independent variable is called the dependent variable. This goes on the
y-axis. For example if you put varying volumes of a protein solution in a series of test
tubes and then measure the absorbance, the variable you control is the quantity of protein,
so it goes on the x-axis. The absorbance is what changes as you change the protein
quantity, so it goes on the y-axis. Always use graph paper when drawing graphs.
UNITS AND EXPONENTS

Assume you did a protein determination and recorded the following data:
Protein content (mg) Absorbance reading at X nm

0.001 0.05
0.003 0.16
0.005 0.225
0.007 0.33
0.008 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.
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 1g then that scale must be
maintained.
17
LABEL AXES
Ensure that both axes are clearly labelled. Axes titles should always include the name of
the variable as well as the unit in which the variable is measured 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.
DRAWING THE LINE

To make a graph you must know something about the system and what you expect to see.
For example, in a protein assay, you expect a straight line. You must then draw the best
straight line possible through the points. This can be achieved through a computer
program (which draws the line for you) or by using a calculator with a statistics function
to tell you the slope of the line, or even by drawing it yourself. Never simply connect the
dots!
If the points on the scatter graph lie within a narrow band, i.e. there is a good correlation
between the two aspects of data, it may be possible to draw a line of best fit that shows
graphically the trend of the correlation. The line of best fit is an approximation or
estimate and it usually goes through the mean of each set of data that you draw. See
Figure 1 below. Note the line of best fit does not necessarily have to pass through the
origin, nor any of the points actually plotted. In biochemistry, however, especially in the
case of calibration curves, the line must pass through the origin.
When the relationship is expected to be linear, the best line is determined by linear
regression. This is a mathematical process in which the line is placed to minimise the
area between the points and the line. This is also called the least squares method.
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.
18
BAD DATA POINTS

You are the one to decide when you have a bad data point. The computer does not make
this decision for you. Table 1 shows a data series that has been plotted on the graph
shown in Figure 2. Notice that 5 of the 6 points fall perfectly on a straight line. However
one point (30, 0.05) is way off. Your first action should be to redo the 30-μg tube rather
than report data of that calibre. If that is not possible, simply draw the line through the 5
“perfect” points and ignore the bad point.
Table 1: Absorbance vs. mg Protein

Protein content (mg) Absorbance at Xnm
0 0.00
10 0.10
20 0.20
30 0.05
40 0.40
50 0.50
0.6
Absorbance @ 750 nm
0.5
0.4
Figure 2. Graph with a bad data
0.3 point
0.2
0.1
0
0 20 40 60
Protein content (ug)
USE THE WHOLE GRAPH

It is best to use as much of the graph as possible. Also, the closer the line is to a 45˚
angle, the more accurate you will be when you interpolate an unknown from it. DO NOT
extrapolate the line beyond the last data point especially in the case of calibration
curves. Refer to Beer-Lambert’s Law and the section on Spectrophotometry.
TITLE:
Your graph must be numbered and have a descriptive title. For example, Graph #1:
Protein Calibration Curve.
19
GRAPHING WITH COMPUTERS
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. Enter your data as shown in Figure 3, which shows what the spreadsheet would
look like. 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. You may also include column labels.
Table 2: Absorbance versus mg Protein for the Lowry

Assay
Protein content (mg) Absorbance at X nm
0 0.00
10 0.10
20 0.21
30 0.29
40 0.40
50 0.52
20
Figure 3. Excel spreadsheet.
Creating the Graph

Once you have loaded the data into the spreadsheet, you are ready to create the graph.
First you need to select the data that will be put into the graph, so click on the upper
leftmost cell and drag down and over until all of your data is highlighted. Then you click
on the wizard button. The first dialog box gives you a choice of chart types, such as bar
graph, pie graph, scatter plot and so on. Normally you will want to do the XY scatter plot,
so this will be used as the example.
There is a potential danger in using the computer to make the graph! The default
value for many programs would yield straight lines connecting the dots. THIS IS
WRONG IN BIOCHEMISTRY!
You want the best-fit line of some type. In your case it would be a linear regression line.
Choose the option XY scatter that does not attempt to put any line over the points. The
lines will be put in later. Click on “Next” to continue the dialog. The next box (box 2 of
4) asks about chart source data. One you have correctly selected the data from your
column, you can go on to the following box by clicking on the “Next”
icon. This brings you to box 3 of 4 in the dialog process. This is the chart-formatting step
where you decide how you want the graph to look. There is a “title: tab you can select
and then add in the graph title and the title of both axes. There is a “gridline” tab that
determines the number of visible gridlines. The forth 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.
21
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.
Remember to always select for a graph that goes through the origin when plotting
calibration curves.
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.
G. SPECTROPHOTOMETRY
Absorption of Light
Almost all biochemical experiments will use the spectrophotometer to measure the
amount of a substance in solution. Spectrophotometry is the study of the interaction of
electromagnetic radiation with molecules, atoms or ions.
Light or electromagnetic radiation has a wave and particle nature. The wavelength, λ, of
light is the distance between adjacent peaks in the wave. The frequency, v, is the number
of waves passing a fixed point per unit time. The parameters can be further defined by the
equation:
λ = c/v
Where c is the speed of light.
Photons of different wavelength have different energies. These energies can be calculated
by the equation:
E = hc/λ = hv
Where h is Planck’s constant. Therefore the longer the wavelength, the less energy the
light has and vice versa. Figure 4 shows the relationship between the wavelength of light
and the common types of electromagnetic radiation. As you can see, those regions where
the wavelength is very short correspond to the types of radiation that you know are
powerful and often harmful, such as X rays, γ-rays and ultraviolet radiation.
Most compounds have a certain characteristic wavelength or wavelengths of light that

they absorb. A solution will look green to us because green light (blue and yellow) is
transmitted while the red light is absorbed.
A solution may contain many compounds that absorb at many different wavelengths but
if a compound we are interested in absorbs at a unique wavelength, we can determine its
concentration even in a solution of other compounds.
22
Fig. 4. The
Electromagnetic spectrum
Beer-Lambert’s Law
If a ray of monochromatic light (l wavelength) of initial intensity I0 passes through a
solution, some of the light may be absorbed, so that the transmitted light I is less than I0.
The ratio of intensities I/I0 is called the transmittance and is dependent on several
factors, which are listed below:
1. If the concentration, c, of the absorbing solution increases, then the transmittance

will decrease
2. If the pathlength, l, that the light must travel through increases, the transmittance
will decrease.
3. If the nature of the substance changes or another substance that absorbs more
strongly is used, then the transmittance will change. The nature of the substance is
reflected in ε, the extinction coefficient, also called the absorptivity constant.
An equation can be written that incorporates these ideas:
log I0/I = εlc
Where: I0 = intensity of incident light, I = intensity of transmitted light, ε =

extinction coefficient, L = path length through solution, c = concentration of
absorbing solution
The log I0/I is usually called the absorbance and is abbreviated A. This law:
A = εlc
is called the Beer-Lambert law (Beer’s law).
23
Some important points to note
1. Absorbance, A, has no units. It is just a number that you read off of the
spectrophotometer. The wavelength is often specified along with the absorbance,
such as A540 = 0.3.
2. The extinction coefficient, ε, is the absorbance of a unit solution and has units of
reciprocal concentration and path length. The most common ε values recorded are
for a path length of 1 cm and a 1M solution. Therefore the expression ε600 =
4000M-1 cm-1 means that a 1 M solution would have an absorbance at 600nm of
4000 if you use a cuvette with a diameter of 1 cm.
3. Remember that the pathlength, l, is usually in cm and if not specified is assumed

to be 1 cm.
4. The concentration, c, will have units that are the reciprocal of the units of ε.
5. You need at least 3mL in a cuvette in order to accurately read it with the Genesys
Spectronic 20 spectrophotometer.
6. Many things can interfere with your use of the spectrophotometer. If the cuvette is
smudged or scratched, light will be scattered by the tube rather than absorbed by
the solution. The light will not penetrate the solution if less than 3mL are in the
cuvette.
If a substance obeys the Beer-Lambert law, then a plot of A vs. c is straight. More often,
however, the line is curved.
Calibration Curves
The concentration of unknown solutions can be determined if you know the extinction
coefficient and if you know that the system obeys Beer’s law at that concentration.
When these things are not known, which is a common occurence, a calibration curve is
prepared. A calibration curve is a plot of absorbance (A) at a particular wavelength vs. a
varying amount of substance of known concentration (your standard solution). Then, an
unknown substance can be determined from the graph. The Beer-Lambert Law often does
NOT hold at high concentrations. A common reason is the depletion of one of the
reagents necessary for colour production. Thus reading should always be taken in the
region where ALL reagents are in EXCESS. It is for this reason that assay mixtures that
are too “dark” (i.e. offscale) must NOT be DILUTED. The original unknown solution
should be diluted before it is reacted and colour is developed.
If you have a compound X of concentration 0.5M in a phosphate buffer, pH 7.0, and the
absorbance readings are as follows for different amounts of compound X,
24
Tube # Volume of Absorbance Corrected

compound X reading at 540nm Absorbance
used (mL)
1 0 0.05 0.00
2 1 0.15 0.10
3 2 0.25 0.20
4 3 0.35 0.30
5 4 0.45 0.40
6 5 0.55 0.50
then to determine the amount of sample X if the absorbance equals 0.3, you must
understand what you have done.
Notice Tube 1 has an absorbance of 0.05 but observe closely and you would realize that
tube 1 does not contain any of the compound we are measuring. Thus tube 1 is called the
reagent blank. A reagent blank is a control in which everything is added EXCEPT
the substance, which is being tested. So, tube 1 is the reagent blank and it has an
absorbance. However, we want a calibration curve that passes through the origin. There
are two ways to achieve a graph that passes through the origin. The first is to subtract the
absorbance (in this e.g. 0.05) from the absorbance reading obtained for each tube. This
gives the data given in the last column, corrected absorbance. This method is tedious and
not necessary given the use of modern spectrophotometers.
The second method is to zero the spectrophotometer with tube 1. That way, the
subtraction is done automatically. You will use this method. Therefore you will always
plot corrected curves.
A CALIBRATION CURVE IS A STRAIGHT LINE THAT PASSES THROUGH

THE ORGIN!
Essential Information
To make use of a calibration curve, you set up a series of tubes varying amounts of
substance of known concentration in them. After measuring the absorbance, plot the
curve (remember it is actually a straight line through the origin). Always plot
Absorbance at the particular wavelenght vs. amount of compound (mg, g, micromoles
etc). Once you have drawn your curve, you can use it to determine the concentration of
your unknown. The absorbance value of the unknown is read on the spectrophotometer
and should fall within the line of your curve, preferably within the linear region
(remember in reality, the line is curved at higher concentrations). Do not extrapolate
your line beyond the highest concentration you have. Once you have determined the
amount of your unknown you can work back to determine its concentration.
25

Lab Manual General Guidelines

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BIOL 2360 Biochemistry II

THE OBJECTIVES OF THE BIOCHEMISTRY LABORATORY INCLUDE:

You are assisted in meeting these objectives by the following:

THINGS YOU SHOULD NEVER DO

2. Mouth pipetting is prohibited. Mechanical pipettes are provided.

3. DO NOT USE MORE CHEMICALS THAN YOU NEED. Chemicals are

LAB PREPARATION & REPORT SUBMISSION

LAB REPORT SUBMISSION

BIOCHEMISTRY LAB REPORTS SHOULD BE TYPED (PREFERABLY) ON

NAME: LAB PARTNER:

ID# DATE (of lab session):

Theory: The theory should pertain to the objectives of the lab. It is a

Procedure: The procedure must be presented in narrative style in the PAST

The discussion should:

1. Answer the questions raised in your Aims/Objectives.

FAILURE TO COMPLY WITH THE ABOVE INSTRUCTIONS WILL RESULT

NB Plagiarism is treated very harshly (whether the source of materials

General Guidelines For A Good Lab Report

 Legible, justified report

B. CITATION OF REFERENCES FOR THE BIOLOGICAL SCIENCES

AUTHOR –DATE SYSTEM

MAKING A CITATION WITHIN THE TEXT:

CITING REFERENCES IN A BIBLIOGRAPHY:

The following lists examples of citations from different sources.

2. Reference to a chapter in a book:

3. Reference to a journal (periodical) article:

4. Reference to an internet source:

 Citing a professional site

LAB SKILLS ASSESSMENT SHEET

Lab Etiquette & Courtesy

Accuracy & Understanding

Statistics and scientific measurements

(i) The extent of the variation about the average.

It should be noted that in biochemical analyses variability in results usually originates

Arithmetic mean (Xavg) = X1 + X2 + X3 + ----Xn = ∑Xi

A second type of statistical measurement is a measure of dispersion. These usually

Consider the following example:

Mean deviation = ∑ |xi – xavg|

Percent deviation = Mean deviation x 100

Variance = σ2 = ∑ (xi – xavg)2

Standard deviation = σ = √σ2

Table 1.1 Basic SI Units

Table 1.2 Prefixes for Multiple Units

13  39g/mol = 0.33 mol

0.333mol  0.5L = 0.666 mol/L = 0.67 M

To use the formula, you need 3 of 4 variables.

C1 = initial concentration, which in this case is 0.2M

DRAWING GRAPHS BY HAND

UNITS AND EXPONENTS

Protein content (mg) Absorbance reading at X nm

PLOTTING DATA POINTS

DRAWING THE LINE

BAD DATA POINTS

Table 1: Absorbance vs. mg Protein

Protein content (ug)

USE THE WHOLE GRAPH

GRAPHING WITH COMPUTERS

Loading the Spreadsheet

Table 2: Absorbance versus mg Protein for the Lowry

Figure 3. Excel spreadsheet.

Creating the Graph

Most compounds have a certain characteristic wavelength or wavelengths of light that

1. If the concentration, c, of the absorbing solution increases, then the transmittance

log I0/I = εlc