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Concepts in Biotechnology & Genetic
Engineering
A level/CAPE Student Workshop
Laboratory Manual
Name______________________
©December 2012, The Biotechnology Centre, UWI
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Schedule of Activities
Students’ Workshop On Concepts In Biotechnology & Genetic Engineering
Host: The Biotechnology Centre, UWI, Mona.
Venue: The Inter-Faculty Lecture Theatre & the Molecular Biology building
December 17-19, 2012
Day 1
8:30-9:00
9:00-9:50
Registration
Lecture 1 (The Importance of Biotechnology – Melaine Randle, senior lab technician
at The Biotechnology Centre, UWI)
9:50-10:00
Workshop objectives (Dr. Marcia Roye, Lecturer in Biotechnology, Biotechnology
Centre, UWI)
10:00-10:30 Break
10:30-11:30 Lecture 2 (Principles of Recombinant DNA Technology - Dr. Roye)
11:30-12:00 Rap session
12:00-2:00
Lunch
2:00-4:00 pm Lab session (DNA extraction - Dr. Roye, Darshan and lab demonstrators)
Day 2
8:30-9:00
9:00-10.00
10:00-11:00
11:00-12:00
12:00-1:00
1:00-3:00
Day 3
8:30-9:00
9:00-10:00
10:00-10:30
10:30-11:30
11:30-12:00
Recap of Day 1
Lecture 3 (Principles of Recombinant DNA Technology - Dr. Roye)
Lab session (Digestion with restriction enzymes to generate a DNA fingerprint- Dr.
Roye, Darshan and lab demonstrators)
Lecture 4 (Principles Underlying the Production of Transgenic Plants- Dr. Natala
Burnett, Lecturer at UWI Mona)
Lunch
Lab Session (Agarose gel electrophoresis of restricted DNA- Dr. Roye, Darshan and
lab demonstrators)
Recap of day 2
Lecture 5 (Principles Underlying the Production of Transgenic Animals- Dr. Aneisha
Collins-Fairclough, Lecturer at UTech).
Break
Lab sessions: Discussion of results – Dr. Roye, Darshan and lab demonstrators.
Closing.
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Experiment 1: Plasmid DNA Extraction
After completing this exercise you should be able to:
1. Perform plasmid DNA isolation from a bacterial culture.
Introduction
The aim of this series of experiments is to use DNA extraction, agarose gel electrophoresis and
restriction fragment length polymorphism (RFLP) to identify the plasmid present in different
bacterial cultures of Escherichia coli (or E. coli for short).
Today’s experiment involves extraction of plasmids from bacterial cultures. In order to achieve this,
the cells are first harvested by centrifugation, lysed and the plasmid DNA is isolated by a series of
precipitation and centrifugation steps.
What are plasmids?
Plasmids are extra-chromosomal, self-replicating, circular (i.e. covalently closed; the term circular
does not indicate actual geometry/shape) DNA molecules. These self-replicating molecules are found in
a wide variety of bacterial species and play an integral role in bacterial adaptation and function.
Plasmids serve as important tools in the study of molecular biology and biotechnology. Plasmid
genes usually give bacteria a selective advantage under some conditions. Gene products encoded by
plasmids include proteins that cause infection in higher organisms, enzymes which allow for the
utilization of unusual carbon sources such as toluene, resistance to heavy metal toxicity and
antibiotics
Plasmid extraction
Plasmids are self-replicating genetic elements that can be genetically engineered to produce high
quality cloning vectors. As cloning vectors, plasmids are useful in biotechnology. In order to be
utilised, plasmid DNA first has to be extracted. This is usually done by alkaline lysis. This procedure
varies among different protocols in that the reagents used in each protocol for the same purpose
may differ, for example precipitation of DNA may be achieved with the use of ethanol or
isopropanol. However there are three basic steps that are common in each protocol, these are;
growth of the bacterial culture, harvesting and lysis of the bacteria, and isolation and purification of
the plasmid DNA. Both the quality and quantity of DNA obtained is dependent on the protocol
used.
You will be provided with a culture of cells labeled with a letter code “X” or “Y” representing
cultures of E. coli cells bearing either one of two plasmids: pCR2.1 or pTrc99A. However you will
not be told which plasmid is present in the culture provided as it is the aim of Experiment 2 to
determine the identity of the plasmid present in your culture of E. coli cells. Each letter code is also
followed by a number, for example “X2” or “Y3”; the combined letter and numerical is unique to your
tube of cells; please ensure that you record this code.
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Materials
Solution 1 (25mM Tris pH 8.0, 10mM EDTA, 50 mM glucose)
Solution 2 (0.2 M NaOH, 1% SDS)
Solution 3 (3 M potassium acetate, 5 M acetic acid)
Isopropanol
70% ethanol
RNAse in TE (100 ug/ml)
microfuge tubes and pipettes.
Procedure
BEFORE YOU BEGIN, PLEASE RECORD THE CODE WRITTEN ON THE TUBE OF CELLS
PROVIDED.
1. To pellet cells, spin down 1.5 ml of overnight culture in a microfuge tube for 3 minutes at
10,000rpm and discard the supernatant.
2. Completely resuspend the pellet in 100 µl of Solution 1.
3. Add 200 µl Solution 2, mix by gently inverting about 6 times and incubate at rtp for 5
minutes.
4. Add 150 µl Solution 3, mix thoroughly by flicking the tube
5. Centrifuge at 10,000 rpm for 10 min and transfer as much of the supernatant as possible to a
new labeled tube, avoiding as much as possible the white precipitate. (Discard tube with
white precipitate).
6. Add 240 µl of isopropanol, mix thoroughly (by gently inverting 4-6 times) and centrifuge at
10,000 rpm for 10 min.
7. Remove the supernatant from tubes by pipetting [Be careful: the white pellet contains your
plasmid DNA. Try not to dislodge the pellet.] Wash the DNA pellet by adding 500 µl of
70% ethanol.
8. Spin for 5 min at 10,000 rpm and remove by pipetting as much of the ethanol as possible.
9. Dry pellets in a speed vac [laboratory assistants will collect your tubes for the drying].
10. Dissolve dried pellets in 30 µl TE/RNase.
Experiment 2: RFLP and Agarose Gel Electrophoresis
After completing this exercise you should be able to:
1. Perform restriction endonuclease digestion of plasmid DNA
2. Set up and run agarose gel electrophoresis of a DNA digest
3. Analyse results of the electrophoresed digestion products.
Introduction
Restriction endonucleases are DNA-cutting enzymes found in bacteria (and commercially isolated
from them for use). They form an important defence against bacteriophages - viruses that prey on
bacteria by attaching to them and injecting their genetic material into the cell. Bacteriophages use the
bacteria to reproduce themselves, they then rupture their host cell before moving on to infect other
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cells. This is similar to what viruses do in animals - using the cells then destroying or damaging
them. Animals have an immune system to fight viral infection; for bacteria, restriction
endonucleases constitute the antiviral defence. They are often called restriction endonucleases
because they cut within the DNA molecule, although they may also be referred to as restriction
enzymes.
Restriction enzymes are of particular use to molecular biologists because they digest DNA at specific
sites based on the nucleotide sequence.
In nature, plasmids often carry additional genes that help the bacteria to survive, for example
antibiotic resistance genes. Molecular biologists use special plasmids, such as pTrc99A and pCR®2.1,
as vectors to transfer genes from one bacteria to another or to study the expression of cloned genes.
This is important for the study of genes and the proteins they encode.
In Experiment 1 you would have extracted one of two plasmids: pTrc99A or pCR2.1. The identity of
the plasmid was not given to you as it is the aim of this experiment to determine the plasmid’s
identity by restriction digestion with the enzyme PstI.
The size of plasmid pTrc99A is 4176 bp and pCR2.1 is 3931 bp.
Once digestion has been achieved, the fragments will be analysed by agarose gel electrophoresis.
Agarose is a seaweed-derived complex carbohydrate polymer that forms a regular microscopic
mesh-like structure (sieve like gel) on cooling (at about 45°C). Agarose gel concentration can be
varied from 0.5 – 2 % (w/v) agarose in a buffer solution.
The DNA fragments are loaded into a well in the gel at the cathode end and an electric current is
applied. DNA has a net negative charge due to the phosphate molecules in the ‘backbone’ of the
molecule, and so moves towards the anode (the positive end). The distance the DNA moves through
the gel is dependent on its size. Therefore, smaller fragments move further than large ones, thus
allowing us to differentiate fragments of DNA molecules on the basis of the distance they move.
Table 1 Cleavage Sites for Restriction Enzyme, PstI
PLASMID
Restriction site (position in bp)
pTrc99A
313
pCR2.1
311, 1501
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Figure 1 Restriction map of plasmid pTrc99A highlighting the positions of restriction sites of
selected restriction enzymes.
Materials
1. 1.5 ml microfuge tube
2. plasmid DNA 1g/l
3. Restriction enzyme PstI 2 Units/l
4. 10x Digestion buffer (50 mM NaCl, 10mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol pH
7.9)l
5. Sterile distilled water
6. Pipettors 10, 20 l
7. Pipette tips, 10, 20 l
8. Water bath at 37°
9. 0.7 % Agarose in 0.5 x TBE 0.5 x TBE buffer
10. Loading dye (50 % (v/v) glycerol, 0.5 % (w/v) bromophenol blue)
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11.
12.
13.
14.
Molecular marker, 1 kb DNA ladder
pTrc99A digested with PstI (Standard)
pCR2.1 digested with PstI (Standard)
ethidium bromide staining solution (0.5 g/ml)
Method
1. Put on a pair of latex gloves. Do not handle tubes or pipettors before – it is possible that your
DNA will contaminate the experiment.
2. Wipe bench area with 70% ethanol or 10 % bleach provided.
3. Label one 1.5 ml microfuge tube with the code of the culture you extracted and “digest” (for
example, x3 digest) and add the reagents as indicated below:
Reagent
Plasmid
Digestion buffer
Water
PstI
Total
Volume (l)
4.0
1.5
8.5
1.0
15.0
4. Mix the contents of each tube by slowly pipetting up and down 2-3 times.
5. Take the tubes to the centrifuge where a laboratory assistant will spin down the contents of your
tube
6. Place the tube in the water bath already set to 37°C and let incubate for 2 hrs.
7. After the incubation period has ended, remove tube from incubator and add 3 l loading dye to
your digestion mix (mix well by pipetting slowly up and down 2-3 times).
Experiment 3: Gel Electrophoresis of DNA
After completing this exercise you should be able to:
1. Set up and run agarose gel of DNA digest
2. Analyse results of the electrophoresed agarose gel
What is electrophoresis?
In this experiment we are separating molecules of DNA that we got from restriction digestion.
Agarose gel electrophoresis is the easiest and most common way of separating and analysing DNA.
The purpose of running the gel might be to visualize the DNA, to quantify it or to isolate a
particular band. The DNA is visualized in the gel by staining in a solution of ethidium bromide.
Ethidium bromide binds strongly to DNA by intercalating between the bases of the DNA molecule.
Ethidium bromide is fluorescent it absorbs UV light and transmits the energy as visible orange light
which we can see and photograph.
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What is a Gel?
You may be wondering what exactly a gel is, and what it has to do with agarose. Let's find out by
"making" a gel. Purified agarose is in powdered form, and is insoluble in water (or buffer) at room
temperature. But it dissolves in boiling water. When it starts to cool, it undergoes what is known as
polymerisation. Rather than staying dissolved in the water or coming out of solution, the sugar
monomers crosslink with each other, causing the solution to "gel" into a semi-solid matrix much like
"Jello", only a bit firmer. The more agarose is dissolved in the boiling water, the firmer the gel will
be. While the solution is still hot, we pour it into a mold called a "casting tray" in order for it to
assume the shape we want as it polymerizes.
How are Gels Loaded and Run?
Imagine you are a DNA molecule. If you were inside an agarose gel, your environment would
resemble a very dense spider web. If you are a small fragment, you could easily crawl through the
spaces in between the webs (they are too tough for you to just pull out of the way). However, as the
length of the molecule increases it gets progressively harder to fit through the spaces. If it were a
race between you and another DNA molecule, who would win? Do you think the same would hold
true for any charged molecule? Now it's time to take the DNA we digested in Experiment 2 and load
it on the gel we just prepared.
Preparing and Running Standard Agarose DNA Gels
The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively
simple and include:
 An electrophoresis chamber and power supply
 Gel casting trays, which are available in a variety of sizes
 Sample combs, around which molten agarose is poured to form sample wells in the gel.
 Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE)
 Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall"
into the sample wells, and one or two tracking dyes, which migrate in the gel and allow
monitoring of the migration of the samples through the gel.
 Ethidium bromide, a fluorescent dye used for staining nucleic acids.
 Transilluminator (an ultraviolet lightbox)
Figure 2 Equipment used for agarose gel electrophoresis
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Materials:


A garose dissolved in TBE(generally 0.7-1%)
0.5 X TBE (same buffer as in agarose)
1. To prepare 100 ml of a 0.7% agarose solution, measure 0.7 g agarose into a glass beaker or
flask and add 100 ml 0.5X TBE
2. Microwave or stir on a hot plate until agarose is dissolved and solution is clear.
3. Allow solution to cool to about 55°C before pouring.
4. Prepare gel tray by sealing ends with tape or other custom-made dam.
5. Place comb in gel tray about 1 inch from one end of the tray and position the comb vertically
such that the teeth are about 1-2 mm above the surface of the tray
6. Pour the gel solution into tray to a depth of about 5 mm. Allow gel to solidify (about 20
minutes at room temperature).
7. To run, gently remove the comb, place tray in electrophoresis chamber, and cover (just until
wells are submerged) with electrophoresis buffer (the same buffer used to prepare the
agarose)
8. Excess agarose can be stored at room temperature and re-melted in a microwave.
9. Mix the contents of each tube (DNA and loading dye) by slowly pipetting up and down 2-3
times.
10. The laboratory assistants will show you how to load your samples into the agarose gel.
Note: Position the pipette tip under the buffer, just above the appropriate well, and slowly
release the sample. SLOWLY expel all the liquid by pressing the plunger to the last position on
the pipettor. Do not release the plunger until the tip has been lifted out of the buffer completely.
Be careful not to shake the electrophoresis box.
11. The samples will be subjected to electrophoresis at 100V for 30 min in 0.5 x TBE buffer.
12. After electrophoresis the laboratory assistants will stain the gels by immersing it in a solution
of ethidium bromide, de-stained in distilled water to remove excess ethidium bromide. The
gel is then viewed and photographed under UV light. You will be given a photograph of
your results.
Congratulations! You have now performed RFLP by digesting a piece of DNA with Restriction
Endonucleases, separating the digested fragments by Agarose Gel Electrophoresis on a gel you
poured, and later you will analyze and document your results (Analysis and Interpretation of the
data).
Use the photograph of the molecular weight marker provided below to assign the appropriate
sizes of the bands of the DNA ladder (molecular weight marker) on the photograph of your gel.
Ask for assistance if you run into difficulty. It is important that this is done accurately in order to
measure the sizes of the restriction products of your plasmid.
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Determine the approximate molecular sizes of each band observed for your digested plasmid by
comparison with the DNA ladder.
Fragment
Size (bp)
1. Based on the RFLP data, which plasmid did you isolate? _____________________
2. Draw a restriction map of your isolated plasmid showing the PstI restriction sites. Your map
must indicate the nucleotide position(s) at which the enzyme cut(s), the size of the plasmid and
an appropriate accompanying title.
Title____________________________________________
Figure 3 1 kb DNA Ladder
visualized on a 0.8% agarose gel.