QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
GENERAL INTRODUCTION TO BIOCHEMISTRY PRACTICALS
Biochemistry is a practical subject and instruction in laboratory techniques is an essential requirement for this course.
Practical courses are usually designed to illustrate selected topics covered in lectures, and at the same time provide
experience in the handling of laboratory apparatus. They are also designed to teach the student to observe accurately,
record concisely, and think clearly; all essential attributes of the trained scientist. To derive maximum benefit from a
laboratory session a student must have a sound understanding of the scientific principles involved and a clear idea of the
experimental objectives.
Importance of practical work
Biochemistry is a practical science and you need to develop certain skills that future employers have every right to
expect you to have. In addition, skills gained in the laboratory enhance your confidence and reinforce your theoretical
knowledge. Practical classes are an essential part of your course of study, and your final assessment will be based in
part on your performance in the laboratory. An interested student who makes proper preparation, and who works
carefully and efficiently, will find laboratory work stimulating and rewarding. On the other hand, an unprepared,
uninterested and careless student will find practical classes tedious and will benefit little from them.
Laboratory note books
A laboratory note book must be 'kept' in which each student should record the following for each practical:
(1)
AIM of the practical
(2)
A FLOW DIAGRAM for the practical. If you are unsure of how to create a useful flow diagram consult your
tutor.
(2)
A LIST OF CHEMICALS needed and any solutions which need preparation. The relative molecular masses
of the required chemicals will be provided.
(3)
OUTLINE OF ANY CALCULATIONS, MANIPULATIONS AND PROCEDURES (e.g. weights of
materials needed and actual weighings made).
(4)
Note any INTERESTING OBSERVATIONS.
(5)
Summary of MATERIALS AND METHODS and RESULTS
(6)
All the above must be completed in the notebook before leaving the practical session.
Materials required by the student
A spatula, a notebook and a glass marking pen or crayon is required.
Safety
A chemical laboratory can become a potentially dangerous place if all safety precautions are not stringently obeyed.
Volatile organic solvents and flames mix with devastating results!
NEVER
*
use volatile organic solvents close to open flames or vice versa;
*
taste anything in the laboratory unless specifically requested to do so by an instructor;
*
point an open vessel you are heating in the direction of anyone.
ALWAYS
*
treat apparatus and chemicals with the greatest of care;
*
work carefully and unhurriedly;
*
consult your instructor if you are in any doubt.
Make sure you know the location of the fire extinguishers and first aid facilities. Carelessness is by far the greatest
cause of laboratory accidents. Please take care at all times.
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 1 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
GENERAL INFORMATION ON PRACTICAL REPORTS
Submission & format
Reports must be handed in by 8 am on the Monday after the relevant practical work has finished. Late reports are not
acceptable. If you are unsure of the format of the report discuss it with the lecturer concerned. See the sections on
report writing in these notes. Abstracts must be concise but give all the relevant information regarding results, including
the conditions (read journals articles to see what is required). Pay particular attention to requirements for figures and
tables and also to standard methods of reporting such things as concentrations, dialysis, centrifugation, enzyme units,
spectra, spectroscopic data and buffers. Check also that you are using the correct nomenclature, accepted abbreviations
and prefaces for multiples or submultiples of units and the correct abbreviation of SI units. Marks will be lost if relevant
units are not given or if the conditions, under which the experiment was carried out, are not given.
Neatness
Reports must be typed up and professionally presented.
Results are important
Therefore poor results are not readily accepted.
Assessment
The marks you obtain for your reports will be used towards your class mark.
Plagiarism
Plagiarism is a serious offence that can result in the loss of a DP certificate. All students are expected to
familiarize themselves with the University of Fort Hare’s Policy on Plagiarism:
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 2 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
THE PRACTICAL REPORT
Presentation of your work
The writing has little to do with literary skill but a lot to do with ORGANISATION which should reveal LOGIC,
CLARITY and PRECISION.
Organisation
You must answer five questions
(1)
Is the report worth reading?
(2)
What was the problem?
(3)
How was the problem tackled?
(4)
What did you find?
(5)
What do your findings mean?
ABSTRACT
AIM/INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Title
All words in the title must be chosen with great care using the fewest that adequately describe the work. Please note that
the titles given in your manual may not be the best title for your report. The biggest faults in title writing are (i)
Improper Syntax (ii) Redundant Words and (iii) Lack of Specificity
Remember also that the title is a LABEL and NOT A SENTENCE. Because it is not a sentence it does not require a
full stop nor does it require perfect grammatical construction, e.g. "Endorphin associated with overeating in genetically
obese mice and rats".
The title does not carry ABBREVIATIONS, FORMULAE, PROPRIETARY NAMES or JARGON
Introduction
This should include a brief statement of the relevant theory and briefly summarize the current literature with references.
In addition, it should include a clear description of the aims of the work (without summarizing the work itself).
Materials and Methods
This section is NOT a reproduction the materials and methods instructions in the practical manual. The golden rule here
is that it must carry enough information so that the experiments can be reproduced. This does not mean that you list
everything and then repeat the listed materials during your description. Nor does it mean that you repeat everything you
have done, because, if you are using well known methodology, it is sufficient to tell the reader the method you used and
where it may be obtained by referencing the original authors.
Materials: Purchased materials should have manufacturers name, exact specification (analysed reagent, chemical
reagent grade, technical grade). Synthesised materials should give the method used. Trade names should be avoided,
but this is not always possible, e.g. TRIS is an easier name than Trishydroxymethylaminomethane and its use is so
common that it is now acceptable. Animals, plants and microorganisms should be identified by GENUS, SPECIES and
STRAIN and the SOURCE identified. Any special characteristics, e.g. age, sex, genetic or physiological state, should
be noted.
Methods: Order is chronological if possible but sometimes related methods should be grouped. This is the first place
that SUBHEADINGS appear. Subheadings are very important in that they guide the reader through your work. Try to
match them with subheadings in your Results and even in the Discussion.
Measurements and Analysis: Was the reaction heated? Give the temperature. At what pH did the reaction occur? Give
the value, the buffer type, the molarity or the ionic strength. Questions such as How? How much? How many? How
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 3 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
long? must be precisely answered.
Referenced Methods: Identify the method, e.g. don't say "cells were broken as described (Johnson et al., 1991)" but
rather "cells were broken by ultrasonics as described by Johnson et al. (1991)".
Results
This is the CORE of your work, and it has two ingredients: (1) it should give an overall description of the experiments
providing a big picture WITHOUT repeating experimental detail, and (2) it should give representative, not repetitive,
data. Your results should be meaningful if you report them. If in a group of experiments a number of variables were
tested one at a time then TABULATE those variables that AFFECT the experiment, for that is your data. However, do
not ignore those variables that did not affect your experiments but don't tabulate them, put them in a footnote or in the
text or in the table legend. Statistics in the result section must be meaningful and the correct statistics applied - you may
have to consult a statistician at some stage. The golden rule for Results is CLARITY which is aided by keeping this
section SHORT. Avoid redundancy and DO NOT repeat tables or figures in the text.
Tables: Only essential data or data needed to illustrate or prove a point should be included in tables. Each table should
have a short explanatory title above the table. Experimental details may be outlined in a footnote to the table ONLY if
the details DO NOT appear in Materials and Methods or in another table. DO NOT PRESENT THE SAME DATA
IN BOTH TABULAR and FIGURE FORM. Each column in a table must carry an appropriate heading. When the
use of abbreviations is necessary, follow the recommendations in the "Policy of the Journal and Instructions to Authors"
of the Biochemical Journal. Units in which data are expressed are given at the top of each column and not repeated in
each line. Do not use ditto marks. Always indicate units of measure clearly. If an experimental condition, such as
dosage or concentration is the same for all of the tabulated experiments, this information should be given in a table
footnote and not in a column of identical figures. Do not include more significant digits in the data than are justified by
the accuracy of the determinations. For example the value 5323 implies an accuracy of 1 part in 5000.
Figures: The most important rule for you here is that the legend should contain sufficient experimental detail to permit
the figure to be interpreted WITHOUT reference to the text (unless this material has appeared with another figure or
table or under Materials and Methods). Quantities and units must be indicated clearly alongside the scales of the
ordinate and abscissa. Wherever possible data should be combined into a single figure, i.e. where abscissa and ordinate
are identical.
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 4 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
Discussion
This is often the hardest section to write because it is here that your in depth knowledge about the subject appears. It
becomes doubly difficult for you who are repeating simple techniques, however our expectations will not be too high,
though you should do some background reading. In the discussion you should:
(1)
present the principles, relationships and generalizations shown by your results;
(2)
NOT recapitulate the results;
(3)
point out EXCEPTIONS and LACK OF CORRELATION;
(4)
define unsettled points;
(5)
show how your work and its interpretation agree (or contrast) with previous work;
(6)
discuss theoretical implications or practical applications of your work (if any);
(7)
state your conclusions CONCISELY AND CLEARLY and summarise the evidence for each conclusion.
References : literature cited
Only significant work should be cited. One of the following two referencing systems are commonly used:
Harvard: Name and Year in text, e.g. Smith and Jones (1989) then list alphabetically under references.
Citation Order: number in text then number order in references. Place the citation where it applies and NOT merely at
the end of a sentence.
In listing references for your use note the following:
Journals:
(1) Authors' names - all
(2) Year
(3) Title of Article
(4) Journal title in full
(5) Volume
(6) Pages (inclusive)
[Example: Ungewickell, E. and Gratzer, W.B. (1978). Self-association of human spectrin. Eur. J. Biochem. 88, 379385]
Books:
(1) Authors' names
(2) Year
(3) Title of Book
(4) Title of Chapter
(5) Edition
(6) Editors
(7) Volume
(8) Pages (inclusive)
(9) Publisher
(10) Place of publishing
[Example: Richardson, J.E. and Richardson, D.C. (1989). In Prediction of Protein Structure and the Principles of
Protein Conformation (Fasman, G.D., ed.), pp. 1-98, J.D. Plenum Press, New York]
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 5 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
CRITICAL OUTCOMES:
1. Collect, organize, analyze and critically evaluate information.
2. Organize and manage oneself and one’s activities responsibly and effectively.
3. Identify and solve problems
4. Work effectively as a team.
SPECIFIC OUTCOMES AND SKILLS:
1. Understanding of the properties of nucleic acids and how these are utilized to purify DNA.
2. Ability to identify the specific chemical properties of the reagents used and explain their function
in isolating DNA.
3. Ability to develop and optimize isolation protocols for any biomolecule of interest.
HEALTH AND SAFETY NOTICE:
1. Care should be taken with all biological material and chemicals used.
2. Exercise caution when handling ethidium bromide. Wear gloves at all times.
3. Toxic substances should not be pipetted by mouth.
4. In the event of spillage wash the site with water.
5. All reagents are expensive and must be treated accordingly
(Instructions to students)
The aim of these practicals is to isolate genomic DNA from plants and bacteria, plasmid DNA from
bacteria and to discover the fundamental properties of DNA and the scientific methods required to
isolate it.
Introduction
DNA is the hereditary material of all living organisms and therefore the isolation of DNA is
essential to geneticists and molecular biologists and scientists interested in studying hereditary
diseases. Almost all cells contain DNA, but not all have equal amounts and therefore it is important
to select the source of DNA carefully. In addition many tissues also have high levels of DNAases
that break down DNA to smaller fragments. The source of DNA should therefore contain high
quantities of DNA and low levels of DNAases. Genomic DNA and plasmid DNA are found in the
cytosol of the bacteria and therefore the first step in isolating DNA is the disruption of the cell
membrane. The isolation of DNA from the cell debris, released proteins, lipids and carbohydrates is
achieved by the careful manipulation of specific DNA properties such as size and polarity. This
practical will give you experience in how to take advantage of the physical and chemical properties
of a biomolecule in order to facilitate its purification from a cell environment.
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 6 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
TITLE: Isolation of Plant DNA from Onions
Method:
1. Isolation of DNA
i.
Place 80 ml of water, 10 ml of dishwashing liquid and 10 g of non-iodized salt into a 250
ml beaker.
ii.
Add 50 g of finely grated onion.
iii.
Incubate in a 60oC water bath for exactly 15 min.
iv.
Filter the homogenate through four layers of cheese cloth (filter paper can also be used)
and save the filtered solution, which contains the DNA.
v.
Add 1 g of meat tenderizer to the strained homogenate then let stand for 5 minutes at room
temperature.
vi.
Cool the homogenate to 10oC on ice.
vii.
Very slowly add 50 ml of ice cold isopropanol (ethanol can also be used) by pouring it
down the side of the tilted beaker. It is essential that the isopropanol and homogenate form
separate layers with the homogenate on the bottom.
viii.
Spool out the white stringy DNA that appears at the interface by gently swirling a glass rod
around at the isopropanol/homogenate interface. Always turn the rod in the same
direction. The DNA will look like a blob of mucus on the glass rod.
ix.
Resuspend DNA in 500 µl TE
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 7 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
TITLE: Isolation of Plasmid DNA
Method:
2. Isolation of Plasmid DNA
i.
ii.
iii.
iv.
v.
vi.
vii.
viii.
ix.
x.
xi.
xii.
xiii.
xiv.
xv.
xvi.
Grow a 5 ml overnight culture with antibiotic selection.
Spin down 1.5 ml in a 1.5 ml eppendorf tube (13 000 rpm for 1 min.). discard broth and
add additional 1.5 ml of culture and spin again. Remove supernatant completely (pour off
broth and dab lightly on a paper towel).
Resuspend cell pellet by vortexing in 0.2 ml Solution 1.
Leave at room temperature for 10 minutes.
Add 0.4 ml Solution 2 and mix well by gently inverting the tube several times.
Leave on ice for 10 minutes.
Add 0.3 ml pre-cooled Solution 3, and mix well by gently inverting the tube several times.
Leave on ice for 10 minutes.
Spin at 13 000 rpm for 5 minutes.
Remove 0.9 ml of the supernatant and transfer to a 1.5 ml Eppendorf tube.
Add 0.6 ml isopropanol, mix and leave for 2 minutes at room temperature to precipitate the
plasmid DNA.
Spin at 13 000 rpm for 5 – 10 minutes.
Retain pellet (be careful not to discard the pellet with the supernatant).
Add 0.5 ml 70% ethanol, rinse by inversion, pour off ethanol (be careful pellet is very
loose), dab gently to dry then spin at 13 000 rpm for 2 minutes and remove remaining
ethanol using a pipette.
Air dry for 5 minutes.
Resuspend DNA in 100 µl TE and leave for about 10 minutes at room temperature. Store
at 4°C.
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 8 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
Method:
3. Preparation of a 0.8% agarose gel
i.
Seal the edges of a clean, dry, plastic tray (supplied with the electrophoresis apparatus)
with tape so as to form a mold. Set the mold on a horizontal section of the bench.
ii.
Add 0.4 grams of powdered agarose to 50 ml of TBE buffer in an Erylenmeyer flask.
iii.
Loosely plug the neck of the flask with cotton wool. Heat the slurry in a boiling-water bath
or a microwave oven on full power for 20 - 60 seconds. Note: heat the slurry for the
minimum time required to allow all the grains of agarose to dissolve. Wearing an oven
glove, carefully swirl the flask from time to time to make sure that any grains sticking to
the walls enter the solution.
iv.
Cool the solution to approximately 60oC (just cool enough to hold). Tutor to add ethidium
bromide to a final concentration of 0.5 µg/ml (2.5 µl of a stock solution of 10 mg/ml in
water) and mix thoroughly.
v.
Position the gel comb near one end of the mold and then pour approximately 40 ml of the
warm agarose solution into the mold. The gel should be between 3 mm and 5 mm thick.
Check to see there are no air bubbles under or between the teeth of the comb. Leave your
gel to set (approximately 45 minutes).
CAUTION: Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be
worn when working with solutions or solids that contain this dye. After use, these solutions and
solids should be placed in designated containers (one for solids and one for liquid) from where they
will be sent for incineration.
4. Evaluation of DNA integrity by agarose gel electrophoresis
i.
After the gel is completely set, carefully remove the comb and tape and mount the gel in
the electrophoresis tank.
ii.
Add enough electrophoresis buffer to cover the gel to a depth of about 1 mm.
iii.
In a sterile microcentrifuge tube (1.5 ml), mix a sample of isolated DNA with gel loading
buffer (10 µl of 6 x DNA loading buffer per 50 µl DNA solution). Slowly load 30 µl of
the mixture into the slots of the submerged gel using a micropipette.
iv.
Load a sample of DNA marker in lane 1. This will provide a ladder of DNA bands of
defined sizes for use as molecular size markers for determining the size of your DNA.
v.
Close the lid of the gel tank and attach the leads so that the DNA will migrate towards the
anode (red label). Apply a voltage of 1 - 5 V/cm (measured as the distance between the
electrodes; normally 50 - 100 V). Electrophorese until the bromophenol blue has migrated
three-quarters the length of the gel, approximately 1 hour.
vi.
Turn off the electric current and remove the leads and lid from the gel tank. Examine the
gel using an ultraviolet (UV) illuminator.
vii.
Photograph the gel using the departmental gel documentation system. The tutor will
demonstrate the use of the UV illuminator and gel documentation system.
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 9 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
CAUTION: Ultraviolet radiation is dangerous, particularly to the eyes. To minimize exposure,
make sure that the UV source is adequately shielded and wear protective goggles or a full safety
mask that efficiently blocks UV light. Discard your ethidium bromide solutions and gel as follows:
1. Place your gel into a special bin for toxic waste. If you are unsure, check with a tutor.
DO NOT discard the toxic gel into the regular dustbins.
2. The buffer into the designated bottles. The ethidium
bromide will discarded
appropriately or be removed using 100 mg/ml activated charcoal. If you are unsure,
check with a tutor. DO NOT discard this toxic solution down the drain.
5. Quantitation and purity of isolated DNA
i.
Mix 30 µl of the DNA solution with 600 µl water.
ii.
Set spectrophotometer to a wavelength of 260nm.
iii.
Zero the spectrophotometer using water.
iv.
Measure and record the absorbance of the diluted DNA solution at 260nm (if
absorbance is > 0.6 dilute sample further).
v.
Set spectrophotometer to a wavelength of 280nm.
vi.
Zero the spectrophotometer using water.
vii.
Measure and record the absorbance of the diluted DNA solution at 280nm (if
absorbance is > 0.6 dilute sample further).
viii.
Calculate the concentration and purity of the DNA sample as follows:
[ DNA(ng / µl )]  A260nm  50ng / µl  dilutionfactor
A
PurityofDNA  260
A280
( Pure DNA will give a ratio  1.8 while a ratio <1.8 indicates that the preparation is contaminated
with proteins and aromatic substances)
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 10 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
Typed practical report for Plant Genomic DNA
Type up your results in the following format:
Title, Abstract and Aim, Results (figure of the agarose gel with appropriate legend, all calculations)
and Discussion & References. In the discussion compare and contrast your results to those that you
would expect and to those values stated in the literature. In addition, in your DISCUSSION address
the following questions:
1. Why dishwashing liquid was added to the initial solution? What commercial reagent can be
used in place of dishwashing liquid?
2. Why is it necessary to add salt to the solution when isolating DNA?
3. What is the function of meat tenderiser and what commercial reagent is normally used in its
place during DNA isolation protocols?
4. Discuss the integrity of the purified DNA and what changes can be made to the above
isolation protocol to ensure intact DNA.
Typed practical report for plasmid DNA isolation
Type up your results in the following format:
Title, Abstract and Aim, Results (figure of the agarose gel with appropriate legend, all calculations)
and Discussion & References. In the discussion compare and contrast your results to those that you
would expect and to those values stated in the literature. In addition, in your DISCUSSION address
the following questions:
1.
2.
3.
4.
5.
Why glucose and EDTA were added to the initial solution?
Why is it necessary to add NaOH and SDS to the second solution when isolating DNA?
Why was K-acetate and acetic acid added to the third solution.
What is the role of isopropanol?
Discuss the integrity of the purified DNA and what changes can be made to the above
isolation protocol to ensure intact DNA.
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 11 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
PRACTICAL ASSESSMENT MECHANISM
CRITERIA:
1.Attendance of practical and participation
2.Completion of practical report
MARK ALLOCATION AND BREAKDOWN:
Title, Abstract and Aim
10
Results:
30
Report on the yield and purity of your DNA using the in-gel and spectrophotometric
methods. Show all calculations and figures.
Discussion:
50
Discuss the function of each of the reagents used in the practical and explain how the
specific reagent performs this function with particular reference to the questions posed in
“Typed practical report”. Include an in depth discussion comparing and contrasting your
yields and purity with those cited in literature.
References & Citations:
10
Correct citation of literature in discussion (5)
Correct listing of references.
(5)
[Total: 100]
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 12 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
TITLE: The Polymerase Chain Reaction (PCR)
INTRODUCTION
The polymerase chain reaction (PCR) has revolutionized how molecular studies are approached and
what questions are asked. The theoretical basis of PCR was first described by Kleppe in 1971, and
then developed by Mullis in 1986 into a technique that could be used to generate large amounts of
single copy genes from genomic DNA (Mullis and Faloona (1987) Methods Enzymol 155, 335350.). The reaction is based on the annealing and extension of two oligonucleotide primers that
flank the target region in double-stranded DNA. The DNA is denatured and annealing of the primers
is allowed such that extension from each 3' hydroxyl end is directed toward the other. The annealed
primers are then extended on the template strand with a DNA polymerase. The three steps of
denaturation, annealing, and extension represent a single PCR cycle. If the newly synthesized
strands extend to or beyond the region complementary to the other primer, it can serve as a primer
binding site and template for subsequent primer extension reactions. Consequently, repeated cycles
of denaturation, annealing and extension result in the exponential accumulation of a discrete
fragment whose termini are defined by the 5' ends of the primers. The discovery of the thermostable
Taq DNA polymerase from the thermophilic bacterium Thermus aquaticus has transformed the PCR
by allowing the development of simple automated thermal cycling devices for carrying out the
amplification reaction in a single tube containing the necessary reagents.
If nucleotide sequence information is available, amplification of DNA by PCR allows the research
scientist to conveniently make many copies (clones) of a segment of DNA of interest. This cloned
segment of DNA may be further manipulated in number of ways such as (i) the production of a
DNA probe for southern hybridization experiments, (ii) or insertion into a suitable expression vector
for expression studies.
Method:
SETTING UP PCR REACTIONS
This practical will be a demonstration practical only.
Setting up the reaction mixture
Before the start of the practical, the tutor must ensure that there are sufficient stocks of Taq
polymerase buffer, Taq polymerase, dNTPs, primers and template. The tutor will set up a reaction
tube (0.2 ml tube) using the required reagents and volumes listed below in Table 2 and a suitable
pipette with sterile tips. Ensure that sterile technique is used throughout and in particular that a
fresh sterile tip is used for each component. The tutor will demonstrate how to pipette in a manner
that prevents carry-over on the outside of the tip and that ensures aerosols are not produced.
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 13 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
Table 1: Recommended Reaction Volumes and Final Concentrations of the GoTaq PCR Core System
Component
Component Volume
Final concentration
MgCl2, 25mM Soln
2.0 – 8.0µl
1.0 – 4.0mM
5X Colorless GoTaq Flexi Buffer
OR 5X Green GoTaq Flexi Buffer
PCR Nucleotide Mix, 10mM each
10µl
1.0X
1µl
200µM each
Forward primer 5 – 50pmol
0.1 – 1.0µl
0.1 – 1 µM
Reverse primer 5 – 50pmol
0.1 – 1.0µl
0.1 – 1 µM
GoTaq DNA Polymerase, 5u/µl
0.25µl
1.25u/50µl
Template DNA
Variable
<0.5µg/50µl
Nuclease-Free water
to a final volume of 50µl
Table 2: Reaction Volumes of the GoTaq PCR Core Systems Components to be used in this Practical
Component
Component Volume
MgCl2, 25mM Soln
3µl
5X Colorless GoTaq Flexi Buffer
OR 5X Green GoTaq Flexi Buffer
PCR Nucleotide Mix, 10mM each
10µl
Forward primer
3.3µl
Reverse primer
3.3µl
GoTaq DNA Polymerase, 5u/µl
0.25µl
1µl
1µl
Template DNA
28.5µl
Nuclease-free water
50µl
Total
Table 3: Thermal Cycling Guidelines for PCR Amplification
Step
Temperature
Time
Number of Cycles
Initial Denaturation
95ºC
2minutes
1
Denaturation
95ºC
1minute
35
Annealing
42 - 65ºC
1minute
35
Extension
72 ºC
1minute
35
Final Extension
72 ºC
5 minutes
1
4 ºC
Indefinite
1
Soak
Expected size of the DNA – 323bp
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
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Page 14 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
Method:
Analysis of PCR products by agarose gel electrophoresis
(i)
After the gel is completely set, carefully remove the comb and tape and mount the gel in the
electrophoresis tank.
(ii)
Add enough electrophoresis buffer to cover the gel to a depth of about 1 mm.
(iii)
In a sterile microcentrifuge tube (1.5 ml), mix a sample of each PCR product with gel
loading buffer (4 µl of 6 x DNA loading buffer per 20 µl PCR product). Slowly load the
mixture into the slots of the submerged gel using a micropipette.
(iv)
Load a sample of 50 bp DNA ladder. This will provide a ladder of DNA bands of defined
sizes for use as molecular size markers for determining the size of your PCR product.
(v)
Close the lid of the gel tank and attach the leads so that the DNA will migrate towards the
anode (red label). Apply a voltage of 1 - 5 V/cm (measured as the distance between the
electrodes; normally 50 - 100 V). Continue electrophoresis until the bromophenol blue has
migrated three-quarters the length of the gel, approximately 1 hour.
(vi)
Turn off the electric current and remove the leads and lid from the gel tank. Examine the gel
using an ultraviolet (UV) illuminator.
(vii) Photograph the gel using the departmental gel documentation system. The tutor will
demonstrate the use of the UV illuminator and gel documentation system.
CAUTION: Ultraviolet radiation is dangerous, particularly to the eyes. To minimize exposure,
make sure that the UV source is adequately shielded and wear protective goggles or a full safety
mask that efficiently blocks UV light. Discard your ethidium bromide solutions and gel as follows:
1.
2.
Place your gel into a special bin for toxic waste. If you are unsure, check with a tutor. DO
NOT discard the toxic gel into the regular dustbins.
The buffer into the designated bottles. The ethidium bromide will discarded appropriately or
be removed using 100 mg/ml activated charcoal. If you are unsure, check with a tutor. DO
NOT discard this toxic solution down the drain.
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 15 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
PRACTICAL REPORT
Prepare your practical report as follows:
1.
TITLE: A statement that captures the essence of the research reported. Avoid redundant and
repetitive phrases. Below the title give your name, institutional affiliation and address.
2.
INTRODUCTION: A concise review of the background, principles and aims and objectives of
the research. In your introduction, include a brief review of the applications of PCR in research,
medicine, and industry.
3.
EXPERIMENTAL PROCEDURES: A concise description of the materials and methods used.
You should not rewrite the quality assurance manual and you should not simply reference the
quality assurance manual. You should have a section in which you state the key reagents and
materials used, in a paragraph form not as a list. You should also have a section for each
method, in which you reduce the methods given in the manual to the minimum required to
reproduce the experiment. For example, you will have to convert the description of the PCR
reaction from the technical recipe given in the manual to one or two sentences with final
concentrations so that a trained scientist could easily translated the procedure back to a recipe
that suits their needs and equipment.
4.
RESULTS: Provide a concise statement of your interpretation of the results obtained for each
figure. Do not simply include figures alone. In addition, refer explicitly to each figure as you
state you interpretations. Your figures should be clearly labelled with all axes or lanes labelled
and any units stated. Remember that all figures should be interpretable by the reader without
reference to the text. In your results include the following figures:
(a) A clearly labelled copy of your agarose gel with a well constructed legend.
(b) A standard curve using your Fermentas 50 bp DNA ladder markers by plotting mobility
(mm) against log bp (DNA fragment sizes in bp = visible: 1031, 900, 800, 700, 600,
500, 400, 300, 250, 200, 150, 100, 50)
5.
Use your graph to estimate the size of your PCR product.
6.
DISCUSSION: Evaluate the significance/meaning of your findings including a statement of
any major conclusions. The discussion should relate the findings to those of other
researchers, and provide a critique of the direction of the research and approach, indicating
experiments that could be done to further the work. Your discussion should include a critical
discussion of the PCR protocol used, the reason for using a water control, an explanation of
the thermal cycling parameters used, how successful they were in amplifying the desired
product, and alternative approaches or protocols. In addition, include in your discussion an
evaluation of the advantages and disadvantages of using PCR and Taq polymerase for
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
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QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
cloning a DNA fragment, especially if the DNA is to be used for further research purposes.
7.
REFERENCES: All references should be cited in the text and listed in a reference section
using the Harvard system as outlined in the general instruction for practicals in the
Biochemistry 323 course.
PRACTICAL ASSESSMENT MECHANISM
CRITERIA:
1.
Attendance and participation in the practical.
2.
Practical report.
MARK ALLOCATION AND BREAKDOWN:
The practical report is marked out of 100 according to the following breakdown and ensuring that
all the aspects stipulated above (under PRACTICAL REPORT) have been addressed in each
section:
1.
TITLE:
[5]
2.
INTRODUCTION:
[5]
3.
EXPERIMENTAL PROCEDURES:
[8]
4.
RESULTS:
[12]
Each set of results represented as a figure should be assessed in terms of the accuracy of the
data generated and the appropriateness and professionalism adopted in the presentation of
the results:
Gel figure with gel lanes labelled and any DNA bands of significance indicated and a legend
that explains the various lanes and labelling:
(5)
Standard curve figure with appropriate axes that are clearly labelled and a
suitable legend:
(5)
Text stating the results with reference to the figures:
(2)
5.
DISCUSSION:
[15]
6.
REFERENCES:
[5]
Citation in the body of the report:
(2)
Reference list:
(3)
[50]
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 17 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
TITLE: SDS-PAGE Analysis of E. coli protein extract
SPECIFIC OUTCOMES AND SKILLS:
1. Understanding of the theoretical concepts of electrophoresis and its modifications.
2. Practical experience with gel electrophoresis, activity gels and protein staining
3.
Application of electrophoresis to the biochemistry and structure of proteins and enzymes
HEALTH AND SAFETY NOTICE:
Wear gloves at all times. Wear masks while preparing and pouring gels
Caution : Acrylamide and TEMED are neurotoxic.
Introduction
Electrophoresis separates biomolecules on the basis of differential migration in an electrical field. In
polyacrylamide gel electrophoresis (PAGE) separation is effected in a gel medium comprising
cross-linked polyacrylamide. PAGE is an important research tool for the separation and purification
of proteins. In combination with the detergent SDS, PAGE can be further used to delineate the
subunit structure of proteins. Conventionally proteins present in the gel are non-specifically stained
with Coomassie Brilliant Blue dye. The advent of activity gels allows the correlation of proteins
bands to bands with specific enzyme activity.
In this practical the principles of SDS gel electrophoresis, protein staining and activity gels will be
employed to investigate the subunit structure and properties of the enzyme tyrosinase (polyphenol
oxidase; EC 1.14.18.1). This enzyme is responsible for the browning observed in bruised fruit and
vegetables. The selective conversion of DOPA (3,4-dihydroxyphenylalanine) to DOPA-chrome by
tyrosinase can be used to identify the presence of functional enzyme
Method:
1. Preparation of Polyacrylamide Gel
You are equipped with a Mini Protean 3 electrophoretic apparatus.
A. SDS-PAGE 1 X Running Buffer (working solution)
Dilute 100ml of 10 X SDS PAGE Running buffer with 900ml deionised water to give a final buffer
with composition of 25mM Tris and 190mM glycine, pH8.3
B. 15% Resolving Gel
1. Mix, in the following order :
5.0 ml of the 30% acrylamide/bis, 37.5:1 mixture, stock
2.5 ml 1 M Tris-HCl buffer (pH 8.8)
2.4 ml distilled H2O
100 µl 10% SDS
50 µl 10% APS
Manual No:
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Date:August 2009
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QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
5 µl TEMED
Manual No:
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QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
2.Pour this resolving gel between the gel plates (set in the casting stand) with a Pasteur
pipette.
3. Pour a very thin layer of water (200 µl) across the top of the gel and allow to set for
approximately 30 min (Leave remaining solution in beaker so you can see when it has set).
C. 4% Stacking Gel
1. Mix, in the following order:
1.3 ml of the 30% acrylamide/bis, 37.5:1 mixture, stock
2.5 ml 1 M Tris-HCl buffer (pH 6.8)
6.1 ml distilled H2O
100 µl 10% SDS
50 µl 10% APS
20 µl TEMED
2. Remove the water from the surface of resolving gel and pour stacking gel as before (Do not
add water layer to the top).
3. Add a 10-tooth comb and leave to set for ± 20 min [Again, leave beaker with remaining
solution so you can see when it has set].
4. Once set, place the gel plate in the buffer container of the electrophoresis apparatus.
5. Remove the comb and fill the apparatus with 1 X running buffer.
2. Sample Loading
[Plan the steps below such that all samples are loaded at roughly the same time]
a) In a microcentrifuge tube resuspend E. coli protein pellet in 500 µl dissociation buffer. Heat
the solution for 5 min in a boiling water bath. Allow sample to cool to room temperature and
load 20 µl.
b) Add 5 µl of a 1x dissociation buffer to 5 µl Rainbow molecular mass markers (this marker
solution is designated M). Do NOT boil. Load 10 µl in Lane 1.
3. Electrophoresis
Run electrophoresis at 200 V until the dye front is within 1 cm of bottom of gel (about 45 minutes)
[NB: Check with your tutor before switching the power on]
4. Coomassie Staining
After electrophoresis, use the scalpel blade to cut the gel as indicated (down lanes 4 and 7).
Place each strip in a separate appropriately labelled Petri dish.
a) Cover the gel the Coomassie staining solution for 0.5 hr to 1 hr.
b) Destain for at least 2x 20 min in Destain solution I.
c) Destain overnight in Destain solution II.
d) The following day scan the destained gels (tutor to book the geldoc imager).
Manual No:
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Date:August 2009
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QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
PRACTICAL WRITE-UP
1. Data Analysis
a) Draw scalar diagrams of each gel, accurately depicting the position and thickness of each
band and the tracking dye.
b) Calculate and tabulate the Rf values of each band of the protein standard on the SDS-PAGE
gel.
c) Plot a graph of log Mr versus Rf values.
d) Interpret the data. Pay particular attention to the number, position, thickness and staining
characteristics of each band representing the E. coli proteins.
2. Typed practical report
Type up your results in the form of a practical report with a Title, Abstract, Introduction,
Methods & Materials, Results & Discussion, and References (please use the guide to report
writing attached to the course outline).
3. Questions
a) Discuss the function of APS, TEMED, glycerol and bromophenol blue
b) Why is water added to the top of the setting resolving gel?
c) Why do the Tris-HCl buffers for the resolving and stacking gels differ in pH?
d) Explain the difference between SDS-PAGE and Continuous Native PAGE and its
significance in the electrophoretic separation of proteins.
PRACTICAL ASSESSMENT MECHANISM
CRITERIA:
1.
Attendance of practical and participation
2.
Accuracy of experimental data, indicating precision of practical work
3.
Understanding of electrophoresis, activity gels and protein staining
4.
Correct analysis of the structure and properties of the subunits of tyrosinase
MARK ALLOCATION AND BREAKDOWN:
Title, Abstract, Introduction, Methods & Materials, References
Results:
Accuracy and precision of experimental data
(correct number, position, thickness and Rf values of protein bands;
well constructed graph of log Mr versus Rf values; correct correlation
of bands between protein staining and activity gels)
Discussion:
Correctness and appropriateness of critical comment and analysis
Correct answers to set questions
Overall quality, conciseness and presentation of write-up
20
25
25
20
10
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 21 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
Total marks
100
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 22 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
PRAC REQUIREMENTS PER PAIR OF STUDENTS
(Instructions to technical staff)
PLANT GENOMIC DNA ISOLATION:
Equipment
Beakers, 250 ml
Ice bath
Pasteur disposable long pipettes
Thermometers
Water bath, 65 o C
Spectrophotometer (UV/Vis)
UV cuvettes
Apparatus for horizontal agarose gel electrophoresis; gel cradle, comb, tank, gel tank cover and
electrode leads.
Power pack (0-250 Voltage supply).
P20 and P200 Pipetteman
Ice bucket with ice
Chemicals
10 ml Sunlight dishwashing liquid
50 ml Isopropanol
1 g Robertson’s Meat tenderizer powder
80 ml Water
250ml TAE
1 ml TE
1 ml 6x DNA loading solution (0.25% bromophenol blue, 40% sucrose in water).
General Requirements
Cheese cloth
Onions
1 x yellow tip box of yellow tips (2-200µl capacity), sterilized by autoclaving
The following should be available for all groups (amounts are total needed for >12 groups).
1. Microwave, cotton wool, and a pair of oven gloves.
2. 100 g agarose (Whitehead Scientific cat # D1 LE) should be available at the balances.
3. Tape (2.5 cm thick for sealing the agarose cradles)
4. 10 ml of ethidium bromide (Sigma cat # E8751; 10 mg/ml in water) stored in a dark bottle.
5. Ethidium bromide liquid waste bottles (5 L winchester bottles).
6. Ethidium bromide solid waste bins in gel documentation room should be cleared.
7. Box of medium latex gloves and a box of small latex gloves.
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
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QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
SPECIAL METHODS, INSTRUCTIONS, RECIPES
6x DNA loading solution
0.25% bromophenol blue (Sigma cat # B-5525), 40% sucrose in water
Prepare 100 ml of this solution. Weigh out 0.25 g bromophenol blue and 40 g of sucrose and
dissolve in 50 ml of distilled water. Make the volume up to 100 ml with distilled water and store at
4oC. This solution will keep for future practicals and should be stored for the following year.
Dispense 1 ml per group.
1 x TE buffer
Add 1 ml 1 M Tris (pH 7.6) and 0.2 ml 0.5 M Na2EDTA (pH 8.0) to 80 ml water. Mix and adjust
volume to 100 ml with water and sterilize by autoclaving. Store at room temperature.
50 x TAE buffer
Dissolve 242 g Tris in 500 ml water. Add 100 ml 0.5 M Na2EDTA and 57.1 ml glacial acetic acid.
Adjust volume to 1 litre with water. Store at room temperature.
1 x TAE buffer
Add 5 ml of 50 x TAE buffer to 245 ml water and mix.
Ethidium bromide (Sigma cat # E8751; 10 mg/ml)
Prepare 10 ml of this solution. Weigh out 0.1 g of ethidium bromide and dissolve it in 10 ml of
distilled water by stirring overnight. Store at 4oC in a brown bottle. This solution will be enough for
many practicals and should be placed out on the day of the practical, but must be placed in the
fridge after each practical and reused the following year.
CAUTION: Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be
worn when working with solutions or solids that contain this dye. Once you have finished preparing
this solution, thoroughly wash all spatulas, weighing boats etc that have been exposed to the dye and
that may be reused for other purposes. Place all other disposables exposed to the dye in the ethidium
bromide solid waste bin in the gel documentation room.
λ DNA molecular marker (Promega cat # D1501)
Prepare 200 µl of this marker. Combine 140 µl sterile ultrapure water (ddH2O), 20 µl restriction
buffer (provided by manufacturer), 40 µl (approximately 20 µg) of λ DNA, 40 units of PstI
restriction endonuclease (Promega cat # R6111) and mix. Incubate at 37oC for at least 2 hours. Add
40 ul of 6x DNA loading solution and mix. This solution is enough for 2 practicals and can be
stored at -20oC. Dispense 10 µl per group in a sterile microcentrifuge tube (1.5 ml).
Manual No:
BCH 323P
Prepared by:GB
Date:August 2009
Issue: 1
Revision: 2
Page 24 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
PLASMID DNA ISOLATION
Solution 1 (10 x stock) (Sterile stocks)
Stock solution
1 M Tris-Cl, pH 8
20 % (w/v) Glucose*
0.5 M EDTA pH 8.0
Water
Final Concentration
0.25 M
0.50 M
0.10 M
ml / 100 ml
25.0
45.5
20.0
9.5
*
Do not autoclave this solution once the components have been mixed as the glucose
caramelizes.
Solution 2 (1 x)
Stock solution
10 M NaOH
25 % (w/v) SDS
Water
Final Concentration
0.2 M
1.0 %
ml / 100 ml
2.0
4.0
94.0
NOTE: This solution needs to be made fresh every week.
Stock solution
K-Acetate
Acetic Acid
Water
Final Concentration
3 moles
2 moles
g / 500 ml
147.0
to pH 5.0
Make up to 500.0 ml
NOTE: Dissolve K-acetate in about 250 ml water, pH to 5 with acetic acid and then make up
to 500 ml with water.
Other solutions required: EtBr (10 mg/ml), isopropanol, 70% ethanol and TE.
Manual No:
BCH 323P
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Date:August 2009
Issue: 1
Revision: 2
Page 25 of 28
QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
POLYMERASE CHAIN REACTION
The following should be available:
1. Pipetman, 2-20 µl; Pipetman 20-200 µl.
2. Yellow tips (2-200 µl capacity) in a yellow tip box; sterilized by autoclaving.
3. Microcentrifuge tubes (0.2 ml capacity); sterilized by autoclaving in a small beaker covered with
foil.
4. Ice bucket with ice.
5. Thermocycler
6. Apparatus for horizontal agarose gel electrophoresis; gel cradle, comb, tank, gel tank cover and
electrode leads.
7. Power supply (0-250 Voltage supply).
8. 250 ml of Tris-Borate-EDTA buffer (TBE; 0.045 M Tris, 0.045 M Borate, 0.001 M EDTA, pH
8.3).
9. 1 ml 6x DNA loading solution (0.25% bromophenol blue, 40% sucrose in water).
SDS-PAGE
2 prs gloves
3 x pasteur pipettes + teat
20-200 ml variable control pipettes
yellow tips
2 x 1 ml microcentrifuge tubes
1 x Mini Protean 3 electrophoretic apparatus + accessories:
2 x glass plates, casting apparatus, 2 x 10-tooth combs, power pack & leads
20 ml 30% acrylamide stock
1 ml 10% ammonium persulphate (APS) [Make fresh]
50 µl TEMED (in an microcentrifuge tube)
30 ml Tris-HCl (pH 8.8)
10 ml Tris-HCl (pH 6.8)
100 ml 10 x SDS running buffer
0.5 ml Dissociation sample buffer (in an microcentrifuge tube)
10 µl Rainbow molecular mass marker solution (in an microcentrifuge tube)
2 x petri dishes OR other suitable container for gel staining
50 ml Staining solution
50 ml Destain solution I
50 ml Destain solution II
Microcentrifuge
Manual No:
BCH 323P
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Date:August 2009
Issue: 1
Revision: 2
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QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
GENERAL REQUIREMENTS
(Instructions to technical staff)
25 L distilled water
1 x tip soak / bench
parafilm
scissors
ice
1.5 ml microcentrifuge tubes
1 x boiling water bath
thermometer for water bath
microcentrifuge tube floaters for the water bath (sufficient for 1 microcentrifuge tube/pair)
pyrex dishes
SPECIAL INSTRUCTIONS AND RECIPES
Clean the glass plates of each electrophoretic apparatus with ethanol and acetone prior to use.
Please check that each electrophoretic apparatus is operational (contacts, leads, power packs
etc.), and that the running buffer allows an operating voltage of 100 V.
1.5 M Tris-HCl buffer (pH 8.8):
For 150 ml:
Tris base 27.23 g
Deionised water 80 ml
Adjust to pH 8.8 and bring total volume to 150 ml with deionised water and store at 4°C.
0.5 M Tris-HCl buffer (pH 6.8):
For 100 ml:
Tris 6g
Deionised water 60 ml
Adjust to pH 6.8 and bring total volume to 100 ml with deionised water and store at 4°C.
30 % Acrylamide stock
Purchase BioRad stock 30% Acrylamide/Bis solution, 37.5:1 mixture (30% T, 2.67% C) –
Catalogue number 161-0158, 500 ml)
SDS-PAGE 10 x Running Buffer (stock solution)
For 1000 ml :
Tris 30.3g
glycine 144.0g
SDS 10.0g
Dissolve in water and adjust volume to 1000 ml with deionised water. Do not adjust pH. Store at 4
°C. Final pH should be ~8.3. If pH is not in this range discard and remake the buffer.
Manual No:
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Date:August 2009
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QUALITY ASSURANCE MANUAL
DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY
PRACTICALS: BIOCHEMISTRY
Subject: Information Flow
Course: Biochemistry 323
Year: 2022
Snr Lab Technician: Dr. N.
Nqumla
E. coli extract
1 ml of o/n culture placed in an eppendorf tube, centrifuge for 1 minute at 13 000g. Discard
supernatant and dissolve pellet in 500 µl dissociation buffer.
Dissociation Buffer
3.55 ml distilled water
1.25 ml 0.5 M Tris HCl (pH 6.8)
2.5 ml glycerol
2.0 ml 10% (w/v) SDS
0.2 ml 0.5% (w/v) bromophenol blue
9.5 ml Total volume
Add 50 µl of ß-Mercaptoethanol to 950 µl sample buffer just prior to use. Dilute sample at
least 1:1 with sample buffer. Heat at 95 °C for 5 minutes.
Coomassie Staining solution
For 100 ml:
45 ml methanol
10 ml glacial acetic acid
0.2 g Coomassie Brilliant Blue
45 ml water
Destain Solution I
For 200 ml:
90 ml methanol
20 ml glacial acetic acid
90 ml distilled water
Destain Solution II
For 200 ml:
10 ml methanol
14 ml glacial acetic acid
176 ml distilled water
Protein Molecular Mass Markers
Order information: Amersham catalogue # RPN 756
Dispense in a microcentrifuge tube, 12 µl per pair of students.
Manual No:
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