UDBM2204 Molecular Biology

Session 201505
Experiment 1
Title: Isolation of Plasmid DNA
Plasmids
Plasmids are relatively small, double-stranded, closed-circular DNA molecules
that exist apart from the chromosomes of their hosts. Plasmids are present in a wide
variety of bacterial and fungal species. Naturally occurring plasmids carry one or more
genes. For example, some plasmids carry genes which confer resistance to certain
antibiotics. Some may carry genes that direct the synthesis of enzymes that aid in the
production of bacterial poisons or antibiotics.
However, from the viewpoint of the genetic engineer, the most important property
of plasmids is that they bear a special region of DNA called an origin of replication, or
more simply an origin. This region allows the plasmid to multiply within and semiindependently of its host. It will replicate its own DNA as well as any passenger DNA
that may be attached to it, producing many copies of the recombinant molecule.
In a typical cloning experiment, the circular plasmid DNA is cut once by treating
it with a restriction endonuclease. This converts the circular molecule into a linear one.
Then, a foreign DNA fragment adds on to the ends of the vector with the help of the
enzyme DNA ligase. The ligase creates a circular molecule containing both the plasmid
and its passenger producing a recombinant DNA molecule. Once inside a suitable host,
the plasmid produces many copies of itself as the bacteria themselves grow and
reproduce.
Extraction and Purification of Plasmid DNA
Many methods have been developed to purify plasmids from bacteria and these
methods invariably involved three steps:
Growth of bacterial culture
Harvesting and lysis of the bacteria
Purification of the plasmid DNA
For many years equilibrium centrifugation in CsCl-ethidium bromide gradients
was the method of choice to prepare large amounts of plasmid DNA. However, this
process is time consuming and requires expensive equipment and reagents. Nowadays,
less expensive and faster methods are available to purify smaller plasmids (<15 kb).
These methods rely on differential precipitation, ion-exchange chromatography, or gel
filtration to separate plasmid DNA from cellular nucleic acids.
The alkaline lysis method is by far the most popular because of its simplicity,
inexpensive, and reproducibility. A variety of kits for plasmid purification are also
available from commercial venders. These kits consist of disposable chromatography
columns that are used for batch absorption and elution of plasmid DNA. However, this
convenience comes at a price.
In this practical, students shall perform plasmid extraction from bacterial culture
using the alkaline lysis method and the commercially available column. The quality of
the extracted plasmids will be compared in subsequent experiments.

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A) Minipreparation of Plasmid DNA by Alkaline Lysis Method
This method is simple, relatively low cost, and has given excellent down stream
experiments results. The isolated DNA is suitable for restriction enzyme digestion, in
vitro transcription, DNA subcloning, DNA sequencing, etc.
Materials
1. Alkaline lysis solution I
(50 mM glucose; 25 mM Tris-Cl, pH 8.0; 10 mM EDTA, pH 8.0)
2. Alkaline lysis solution II
[0.2 N NaOH, 1% (w/v) SDS, solution II should be prepared freshly and used at
room temperature)
3. Alkaline lysis solution III
(3 M potassium, 5 M acetate)
4. Ethanol
5. Phenol:chloroform (1:1, v/v)
6. DNase free RNase
7. Microfuge tubes
8. Micropipettes and tips
Methods:
Preparation of Cell
1. Inoculate a single colony or 10 l of previously frozen bacteria containing the
plasmid DNA of interest in 5 ml of LB medium and 50 g/ml of appropriate
antibiotics (e.g., ampicillin), depending on the specific antibiotic-resistant gene
carried by the specific plasmid. Culture the bacteria at 37C overnight with
shaking at 200 rpm.
2. Transfer the overnight culture into microcentrifuge tubes (1.5 ml per tube),
centrifuge at maximum speed for 1 min, and then discard the supernatant.
Lysis of Cells
3. Resuspend the bacterial pellet in 100 l of ice-cold alkaline lysis solution I by
vigorous vortexing (make sure that the bacterial pellet is completely dispersed in
Alkaline lysis solution I. Vortexing the microfuge tubes simultaneously with their
bases touching increases the rate and efficiency with which the bacterial pellets
are resuspended)
4. Add 200 l of freshly prepared Alkaline lysis solution II to each bacterial
suspension. Close the tube tightly, and mix the contents by inverting the tube
rapidly five times. DO NOT vortex! Store the tube on ice for 5 min
5. Add 150 l of ice-cold Alkaline lysis solution III. Close the tube and disperse
Alkaline lysis solution III through the viscous bacterial lysate by inverting the
tube several times. Store the tube on ice for 5 min.
6. Centrifuge the bacterial lysate at maximum speed for 5 min and carefully transfer
the supernatant to a fresh tube.
7. (Optional) Add an equal volume of TE-saturated phenol:chloroform. Mix the
organic and aqueous phases by vortexing for 1 min and then centrifuge the
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emulsion at maximum speed for 10 min. Transfer the aqueous upper layer to a
fresh tube
Recovery of Plasmid DNA
8. Precipitate nucleic acids from the supernatant by adding 2 volumes of 100%
ethanol (or 1 volume of propanol) at room temperature. Mix the solution by
vortexing and then allow the mixture to stand for 2 min at room temperature.
9. Collect the precipitated nucleic acids by centrifugation at maximum speed for 10
min. Carefully decant the supernatant, add 1 ml of 70% ethanol to the pellet and
invert the closed tube several times. Recover the DNA by centrifugation at
maximum speed for 5 min.
10. Remove all of the ethanol by gentle aspiration and dry the plasmid DNA under
vacuum for 15 min or store the open tube at room temperature until the ethanol
has evaporated and no fluid is visible in the tube.
11. Dissolve the plasmid DNA in 50 l of TE (pH 8.0) buffer or sterile deionized
water. Take 1 l to measure the concentration and store the DNA solution at 20C until use.

Reference :
1. Sambrook, J., Russell, D. W., Sambrook, J. (2001). Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory

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Session 201505
Experiment 2
Title: Polymerase Chain Reaction
Introduction
Polymerase Chain Reaction (PCR), invented by Kary B. Mullis, at the Cetus
Corporation, who was awarded the 1993 Nobel Prize for chemistry for PCR, is a
technique to exponentially amplify in vitro a small quantity of a specific nucleotide
sequence.
A PCR reaction consists of the template DNA to be amplified, a pair of
oligonucleotide primers with sequence flanking the region to be amplified, DNA Taq
polymerase, the four deoxynucleoside triphosphates (dNTP), magnesium, and appropriate
buffer and salts.
The reaction is cycled involving template denaturation, primer annealing, and the
extension of the annealed primers by DNA polymerase until enough copies are made for
further analysis. During the PCR process, the product of 1 cycle serves as template in the
next cycle. Amplification of template progress at a rate of 2n, where n is equal to the
number of cycles.
PCR is a very powerful technique and is used in wide range of applications. To
cite only a few, it is used to examine biological evidence in forensic cases, to identify
contaminating microorganisms in food, to diagnose genetic diseases, to map genes to
specific chromosome segments, to detect the presence of a specific DNA in a particular
sample, etc.
In this practical you will use the plasmid DNA extracted in experiment 1 as
template in the PCR reaction to detect the presence or absence of a foreign DNA in that
plasmid. The PCR product can be detected by performing agarose gel electrophoresis.
Materials
1. PCR reaction components
2. Micropipettes and tips
3. Microfuge tubes

Methods
A. Avoiding Contamination
PCR allows the production of more than 10 million copies of a target DNA
sequence from only a few molecules. The sensitivity of this technique means that the
sample should not be contaminated with any other DNA or previously amplified products
(amplicons) that may reside in the laboratory environment. Below are some precaution
steps to avoid contamination:
DNA sample preparation, reaction mixture assemblage and the PCR process, in
addition to the subsequent reaction product analysis, should be performed in
separate areas.
A Laminar Flow Cabinet equipped with a UV lamp is recommended for preparing
the reaction mixture.
Fresh gloves should be worn for DNA purification and each reaction set-up.
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The use of dedicated vessels and positive displacement pipettes or tips with
aerosol filters for both DNA sample and reaction mixture preparation, is strongly
recommended.
The reagents for PCR should be prepared separately and used solely for this
purpose. Autoclaving of all solutions, except dNTPs, primers and Taq DNA
Polymerase is recommended.
Solutions should be aliquoted in small portions and stored in designated PCR
areas. Aliquots should be stored separately from other DNA samples.
Detailed instructions about PCR laboratory setup and maintenance may be
obtained from the instructor.
Therefore, a negative control reaction, omitting template DNA, should always be
performed, to confirm the absence of contamination. A positive control reaction can also
be performed to ensure all the PCR reagents are working.
B. Preparation of Reaction Mixture
To perform several parallel reactions, prepare a master mix containing all the
PCR components except the template DNA in a single tube, which can then be aliquoted
into individual tubes. Template DNA solutions are then added. This method of setting
reactions minimizes the possibility of pipetting errors and saves time by reducing the
number of reagent transfers.
Reaction Mixture Set Up
1. Gently vortex and briefly centrifuge all solutions after thawing except for Taq DNA
Polymerase.
2. Label your PCR reaction tubes and a master mix sterile 1.5 ml microfuge.
3. Your instructor will instruct you the amount of each reagent to be added in the PCR
reaction.
4. Calculate the amount of each individual PCR component and prepare a master mix
solution in the sterile 1.5 ml microfuge tube as follow:
Reagent
Sterile deionized water
10X Taq buffer
dNTP mix
Primer I
Primer II
Taq DNA Polymerase
25mM MgCl2
Template DNA

Final
concentration

Quantity for 1
reaction mixture (l)

Quantity for master

mix (l)

1
0.2 mM of each
0.1-1 M
0.1-1 M
1.25 U/50 l
1-4 mM
10 pg-1 g
Total

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5. Gently vortex the sample and briefly centrifuge to collect all drops from walls of
tube.
6. Aliquot the appropriate amount of master mix to each reaction tube.
7. Dilute the PCR template as instructed by the instructor.
8. Add the diluted DNA template into the labeled PCR tubes.
9. Overlay the sample with a drop of mineral oil. This step may be omitted if the
thermal cycler is equipped with a heated lid.
10. Place samples in a thermocycler and start PCR (the amplification parameters will be
provided by the instructor).
11. Record the amplification parameters as follow.
Cycling steps
Initial denaturation
Denaturation
Primer annealing
Extension
Final Extension

Time

Number of cycles

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Session 201505
Experiment 3
Title: DNA Detection by Agarose Gel Electrophoresis
Agarose gel Electrophoresis is based on the principle of separating molecules by
their attraction to an electrical charge. Because DNA contains a negative charge owing to
the phosphate groups linking the deoxyribose backbone, it will migrate towards the
positive pole when placed in an electrical field. Two major types of gel electrophoresis
are use in DNA research, agarose and polyacrylamide. Agarose is a highly purified form
of agar and, when solidified, will make a network through which DNA must move. The
result is that large DNA fragments will migrate more slowly through the agarose,
whereas smaller DNA fragments will migrate more rapidly. These different rates allow
for the separation of a mixture of DNA fragments by their molecular weights. The
molecular weight of a particular DNA can be used to determine the approximate number
of nucleotides in that DNA.
Ethidium Bromide Staining
The most convenient and commonly used method to visualize DNA in agarose
gels is staining with the fluorescent dye ethidium bromide, which contains a tetracyclic
planar group that intercalates between the stacked basses of DNA. Ethidium bromide
binds to DNA with little or no sequence preference. Upon UV radiation, DNA bound
ethidium bromide will display an increased fluorescent yield compare to that of dye in
free solution.
Materials
1. Gel casting and electrophoresis apparatus
2. Agarose
3. Power supply
4. 1X TAE buffer (40mM Tris-acetate, 1mM EDTA)
5. 6X gel loading buffer [0.15%(w/v) bromophenol blue, 9% (w/v) Ficoll 400, 40%
(v/v) glyserol]
6. Ethidium bromide solution (0.5 g/ml in dH2O)
7. 1 kb DNA ladder
8. UV transilluminator
9. Micropipettes and tips

Methods
A. Casting the gel:
1. Make 25 ml of a 1.0% (w/v) solution of agarose in 1 TAE or 1 TBE buffer.
2. Weigh the container with the mixture and record the mass.
3. Heat the mixture to boiling using the microwave oven. Examine the flask and
continue boiling until all agarose is completely dissolved.
4. Weigh the container with the mixture again and add deionized water to
compensate for loss of mass during boiling.
5. Allow the agarose solution to cool for 3-5 minutes at room temperature before
pouring into the gel casting apparatus.
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6. Meanwhile, assemble the gel casting apparatus as instructed.
7. Pour the cooled agarose solution into the casting tray, being careful not to
overflow the tray. Place the comb and leave the gel to cool and solidify.
B. Preparing the samples
1. While the gel is cooling, prepare the DNA samples by adding 1 l of tracking dye
to 5 l of extracted plasmid DNA from Experiment 1, and 5 l of PCR product
from Experiment 2, respectively. Adding tracking dye to the sample will increase
its density so it falls into the well of the gel and provides a visible marker to
monitor the progress of electrophoresis.
2. Also prepare a molecular size standard by mixing 5 l of the 1 kb ladder with 1 l
of tracking dye.
C. Loading and running the gel
1. Carefully remove the comb from the solidified gel by lifting it straight out of the
gel slowly.
2. Place the gel in the electrophoresis tank and fill the buffer reservoir with the same
buffer used to dissolve the agarose powder until the buffer is 1-2 mm deep over
the gel.
3. Carefully pipette each mixture (6 l) into a well in the gel. Observe the
demonstration by the instructor before performing this step. Load one well with
the prepared 1 kb DNA ladder.
4. After all the lanes have been loaded, connect the leads from the power supply to
the gel box. Ensure the gel is oriented correctly (wells at negative [black] end).
DNA in the wells will migrate from cathode to the anode.
5. Set the output level to 80 volts and turn the power on.
6. Turn off the power supply when the tracking dye is approximately 1 cm away
from the anode end of the gel.
D. Staining the DNA in the gel with ethidium bromide.
1. After turning the power off, remove the gel from the gel box and submerge it in
the ethidium bromide staining solution. Allow the gel to stain for 5 minutes.
WARNING: ETHIDIUM BROMIDE IS A MUTAGEN. WEAR GLOVES
WHEN HANDLING IT.
2. Remove the gel and submerge it into water briefly.
E. Photography
1. Place the gel on the transilluminator. Perform the following steps with supervision
from the instructor.
WARNING: THE TRANSILLUMINATOR EMITS SHORT WAVE UV
LIGHT WHICH WILL DAMAGE SKIN AND EYES, DURING PROLONGED
EXPOSURE. BE SURE THAT PROPER SHIELDING IS IN PLACE BEFORE
TURNING ON THE TRANSILLUMINATOR.
2. Turn on the transilluminator (BE SURE THAT PROPER SHIELDING IS IN
PLACE). Turn on the Gel Documentary Image Analyzer and observe the gel on
the screen. Capture a picture of the gel.
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Session 201505
3. Turn off the transilluminator. Remove the gel and discard it into the provided
trash can.

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Session 201505
Experiment 4
Title: Analysis of the topoisomers of plasmid DNA following treatment with a
restriction endonulease or DNA Topoisomerase 1
DNA Supercoiling
The double stranded circular DNA or linear DNA by complexing with proteins,
can have tertiary or higher order structure such as supercoiling or supertwisting or
superhelicity. The supercoiling literally means the coiling of a coil. For example: A
telephone cord is typically a coiled wire.
A circular DNA without any superhelical turn is known as relaxed molecule.
Supercoiling of DNA is an important feature of all chromosomes from those of the
smallest viruses to those of eukaryotes. Supercoiling generate more compact structure,
important for packing long DNA molecules into chromosomal structure or into viral
cells.
DNA can be negatively suprecoiled (right handed) or positively supercoiled (left
handed). Negative Supercoiling results from underwinding or unwinding, where as
positive supercoiling results from tighter winding or overwinding of DNA double helix.
A supercoiled DNA molecule is more compact than a relaxed DNA molecule of the same
length. Therefore, supercoiled DNA moves faster than relaxed DNA molecule when they
are centrifuged or electrophoresed. Hence they can be separated by agarose gel
electrophoresis or by equilibrium centrifugation.

Topoisomerases
DNA supercoiling is regulated in every cell that influences many aspects of DNA
metabolism. The normal biological functioning of DNA occurs only if it is in the proper
topological state.
The supercoiling of DNA is controlled by a remarkable groups of enzymes known
as Topoisomerases. They are so named because they alter the topological state (linking
number) of circular DNA but not its covalent structure. Topoisomerases play important
role in processes such as replication & DNA packing. There are two classes of
topoisomerases:
(1)Type 1 Topoisomerases -- act by creating transient single strand breaks in DNA &
change L in increments of 1.
(2)Type 2 Topoisomerases -- act by making transient double strand breaks in DNA &
change "L"in increments of 2.

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Materials:

Plasmid DNA from experiment 2

Deionized or distilled H2O

Calf thymus DNA Topoisomerase I

10 Topoisomerase reaction buffer

10 restriction endonuclease buffer

Restriction endonuclease that single digest the plasmid

Microfuge tubes

Micropipettes and tips

37oC incubator or water bath

Electrophoresis apparatus and power supplies

0.8% agarose in 1X TAE (melted)

1 TAE for electrophoresis units

Ethidium bromide solution

Staining trays

UV transilluminator
Methods:
Treatment of Plasmid with Restriction Endonuclease
1.
Set up the restriction enzyme digestion of plasmid as shown below in a 1.5 ml
microcentrifuge tube (listed in order of addition):
Reaction component
Deionized or distilled H2O
10 reaction buffer
Plasmid DNA
Restriction endonuclease
2.
3.
4.
5.

Tube number
E1
E2
6
5
1
1
3
3
1

Briefly centrifuge the microfuge tube to collect the fluids at the bottom of the tube.
Incubate the tubes for 1 hour at 37 C (in a water bath or incubator). Store reaction
tubes on ice until ready for agarose gel electrophoresis analysis.
Perform agarose gel electrophoresis to analyze the digests.
Obtain a print out of the gel image for result analysis.

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Time Course Treatment of Plasmid with Topoisomerase I
1.
Add five microliters of gel loading dye to each of six microcentrifuge tubes labeled
T0, T2, T5, T10, T20, and T30. Place all the six tubes on ice.
2.
Prepare reaction mixtures as described below in a microcentrifuge tube (listed in
order of addition) and keep the tube on ice.

Reaction component
Deionized or distilled H2O
10 reaction buffer
Plasmid DNA
DNA Topoisomerase I

3.
4.

5.
6.

Volume
35
5
5
5
(Add only after 5 l of the above
reaction mixture removed and mixed
with loading dye in T0 tube)!

Mix the reaction mixtures and incubate the reaction tube at 37C.
Remove a 5 l aliquot at 2, 5, 10, 20 and 30 min of incubation, mix with the loading
dye in the appropriately labeled tube and place on ice. Continue incubating the
reaction tube.
Perform agarose gel electrophoresis to analyze all the samples.
Obtain a print out of the gel image for result analysis.

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Experiment 5
Title: Enzyme synthesis regulation in Escherichia coli
The Lactose Operon
The lactose operon encodes three proteins, LacZ - -galactosidase, LacY - the lactose
permease, and LacA - lactose transacetylase. The operon is repressed by the LacI
repressor whose gene is close to, but not part of the lac operon. Presence of inducer
inactivates the repressor. Lactose itself is not an inducer and only indirectly induces the
lac operon after a small amount has been isomerized to allolactose. This is an isomer of
lactose, generated in a side reaction by the low basal levels of -galactosidase which are
found before induction. In the laboratory, IPTG (isopropyl-thio--D-galactoside) is often
used as inducer. IPTG is not metabolised and is of no use to the cell - it is a gratuitous
inducer.

Catabolite Repression
Presence of a favored carbon source such as glucose prevents use of less favored
substrates such as lactose. Catabolite repression depends largely on the intracellular level
of cyclic AMP. Cyclic AMP is bound by Catabolite Activator Protein (CAP) also known
as cyclic AMP Receptor Protein (CRP). The level of CRP is constant. Transcription of
catabolite sensitive operons such as the lac operon requires binding of CRP-cAMP
complex to the promoter region. This allows RNA polymerase to bind to and transcribe
the operon.

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Session 201505
The regulation of cyclic AMP levels is due mostly to changes in activity of adenylate
cyclase which catalyses the conversion of ATP to cyclic AMP plus inorganic
pyrophosphate. The presence of glucose causes a drop in the activity of adenylate cyclase
and hence a drop in cyclic AMP levels. Glucose must be transported for this to happen,
but it does not need to be broken down and metabolized. Non-metabolizable analogs of
glucose, such as 2-deoxyglucose, cannot be degraded but can be transported and also
cause catabolite repression.

Procedure
A.

Cell Growth

a) Inoculate Lac+ strain of E. coli cells into 5 ml basic medium plus 2% glycerol and
shake overnight at 37C.
b) Approximate 2 hrs before use add 2.5 ml of overnight culture to 50 ml basic medium
plus 2% glycerol.
The idea is to use cells that are somewhat starved and that are in the log phase of growth.
Glycerol is not a good energy source, so the cells are not able to grow as fast
as possible. By diluting the overnight culture and letting it grow for two hours, you allow
the cells to enter the log phase of growth.

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B.

Induction of Enzyme

The synthesis of -galactosidase may be induced using the following procedure. Into a
large size (18 mm) labeled test tube place:
a) 4 ml of starved E. coli cells (1 x 107 cells/ml).
b) 0.2 ml of 0.002 M inducer (LAC, GLU, IPTG, or dH2O)
Put a cap on each tube, place in a 37 C water bath and aerate (shake) for 30 minutes.

C.

Assay for Enzyme

Although ONPG is used to determine whether or not -galactosidase has been

synthesized in the cell, the compound will not quickly pass through a living cell
membrane. Therefore the E. coli must first be treated with a detergent, sodium
desoxycholate, and an organic solvent, toluene, to destroy the selective permeability of
the cell membrane. This treatment, which allows ONPG to enter the cell quickly, also
kills the cells, but does not affect the activity of the enzyme. CAUTION! These
compounds are also toxic to humans.
1. Disruption of Selective Permeability:
To 4.2 ml of an induced E. coli culture add:
1) One drop sodium deoxycholate (1.0 mg/ml)
2) One drop toluene
Cap, place in a 37C water bath and aerate or shake for 10 minutes. This preparation may
be used for enzyme assays. Keep it in an ice bucket and only remove sample when
needed for assays.
2. Enzyme Assay:
Into each small labeled culture tube place:
1) 2.0 ml of 0.1 M sodium phosphate buffer (pH 7)
2) 2.0 ml lysed E. coli preparation
3) 0.2 ml of 0.01 M ONPG (Substrate)
Incubate for 15 minutes at 37 C without shaking. Stop the reaction by adding 0.5 ml 2M
sodium carbonate. This will make the solution alkaline (pH>8) and denature the enzyme.
Read the absorbance at 420nm in a spectrophotometer. If the compound is an inducer,
more enzyme will be formed, more substrate (ONPG) will be converted to a yellow
product and the absorbance will be higher.

Equipment
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37C water bath shaker (or water bath and aeration set-up)
Sterile capped test tubes
Ice bath
Spectrophotometer
Cuvettes
Pipettes (0.2 ml, 1 ml, 5 ml)

Materials and Reagents

E. coli (Lac+)culture
0.002 M lactose
0.002 M glucose
0.002 M IPTG (iso-propyl--thiogalactoside)
0.002 M PBG (phenyl--galactoside)
1.0 mg/ml sodium deoxycholate
Toluene
0.01 M ONPG (ortho-nitrophenyl--galactoside)
0.1 M sodium phosphate buffer, pH 7
2 M sodium carbonate

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Experiment 6
Title: Induction of gene expression
This experiment consists of two components:
A. induction of gene expression
B. protein detection by SDS-PAGE
A. Induction of gene expression in E. coli
Bacterial culture of E. coli bearing a plasmid with a gene regulated by the lac
operon will be used. Addition of IPTG into the culture will trigger the expression of the
gene. To study the expression of the IPTG inducible gene, a time course experiment will
be carried out. The bacterial cells will be pelleted for every 1 hr interval. The expressed
proteins for every 1 hr intervals will be analyzed by SDS-PAGE.
Materials
Over night E. coli culture
LB broth
Isopropyl--D-thiogalactoside (IPTG)
Microfuge tubes
Micropipettes and tips
Methods:
1. Inoculate a pure colony of E. coli with the vector that carries the gene or DNA to
be expressed into 10 ml LB medium containing appropriate antibiotic.
2. After overnight incubation at 37C with shaking at 180 rpm, inoculate 100 l of
the overnight culture into 10 ml of LB medium.
3. Grow the diluted culture at 37C, with vigorous shaking at 220 rpm for 2 to 3 hr.
4. Remove 1 ml of the culture and pellet the cells using a bench top centrifuge
5. Store the pellet in -20C for the next experiment.
6. Add IPTG to a final concentration of 1 mM to the remaining culture.
7. Incubate the culture at 37C, with vigorous shaking at 220 rpm for an additional 3
to 5 hrs.
8. Pellet the cells as described in steps 5 and 6 every 1 hr intervals.

B.

Protein detection by SDS-PAGE

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE is a technique used in biochemistry and molecular biology to separate
proteins according to their size (length of polypeptide chain). SDS works by disrupting
non-covalent bonds in the proteins, thereby denaturing them, causing the molecules to
lose their native shape (conformation). Also, anions of SDS bind to the main peptide
chain to effectively impart a negative charge on the protein that is proportional to the
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mass of that protein (about 1.4 g SDS/g protein). This new negative charge is
significantly greater than the original negative charge of that protein. The electrostatic
repulsion that is created by binding of SDS causes proteins to unfold into a rod-like shape
thereby eliminating differences in shape as a factor for separation in the gel, so that the
distance of migration through the gel is directly related to only the size of the protein
The solution of proteins to be analyzed is first mixed with SDS to denature the
proteins and apply a negative charge to every protein.
Preparations will involve casting of two different layers of acrylamide between
glass plates. The lower layer (separating, or resolving, gel) will be responsible for
actually separating polypeptides by size. The upper layer (stacking gel) will include the
sample wells, and will be of a composition that causes the samples to become
compressed (stacked) in order to have thin bands and correspondingly better resolution
among bands.
Depending on their size, each protein will move differently through the gel
matrix: short proteins will more easily fit through the pores in the gel, while larger ones
will have more difficulty. After a set amount of time (usually a few hours), the proteins
will have differentially migrated based on their size; smaller proteins will have traveled
farther down the gel, while larger ones will have remained closer to the point of origin.
After electrophoresis the separated proteins need to be stained. A commonly used
stain for detecting proteins in polyacrylamide gels is 0.1% Coomassie Blue dye in 50%
methanol, 10% glacial acetic acid. Acidified methanol precipitates the proteins. Staining
is usually done overnight with agitation. The agitation circulates the dye, facilitating
penetration, and helps ensure uniformity of staining.

SDS-PAGE
Materials and Reagents:

Acrylamide/Bis

SDS

SDS-PAGE electrophoresis
apparatus

Resolving gel buffer [1.5 M TrisHCl, pH 8.8]

Stacking gel buffer [1.0 M TrisHCl, pH 6.8]

SDS-PAGE electrophoresis buffer

SDS-PAGE sample loading buffer

(SAB)

Mercaptoethanol
Coomassie Blue stain
Destain solution
N,N,N,Ntetramethylethylenediamine
(TEMED)
Ammonium persulphate (APS)
Microfuge tubes
Micropipettes and tips
Protein standard marker

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Lab Manual 201505
Bachelor of Science (Hons) Microbiology

UDBM2204 Molecular Biology

Session 201505
Methods:
1.
Assemble the glass plates vertically according to the instructions of the
manufacturer.
2.
Prepare the resolving gel in 1.5 M Tris-HCl, pH 8.8 and the stacking gel in 1.0 M
Tris-HCl, pH 6.8 (refer to table below).
3.
Polymerize both gels with 0.1% (w/v) APS and 0.1% (v/v) TEMED one after
another, with the resolving gel at the bottom and the stacking gel on top where the
wells will be formed.
4.
Insert the comb to form the wells.
5.
After the polyacrylamide gel had polymerized, secure the gel sandwich to a vertical
electrophoresis apparatus.
6.
Fill the upper and lower buffer chambers with SDS-PAGE buffer until the top and
bottom ends of the gel sandwich are submerged in the buffer.
7.
Before the protein samples are loaded, flush the wells with SDS-PAGE buffer.
8.
Add 20 l of 1XSAB to the cell pellet and heat the samples for 2-5 minutes at 99C.
9.
Place the Heat-treated samples on ice and load 10 l of each samples into the wells.
10. For protein size reference, load 5 l of Pre-stained SDS-PAGE standard.
11. Perform electrophoresis at a constant voltage of 200 V until the blue dye has
reached the bottom of the gel.
12. After electrophoresis, stain the gel with Coomassie Blue with agitation
13. Destain the gel with destaining solution.

Discontinous SDS-PAGE gel formulation (10ml)

30%
Gel percentage
10% w/v SDS
sdH2O (ml)
Acrylamide/Bis
Gel buffer (ml)
(%)
(ml)
(ml)
5
5.7
1.7
2.5 (stacking)
0.1
10
4.1
3.3
2.5 (resolving)
0.1
For polymerization, 50 l 10% APS and 5 l TEMED were added to the gel solution.
Questions:
1.
Would you expect E. coli Lac- to grow on a medium in which lactose is the only
carbon source? Why?
2.
Although lactose (and thus allolactose) is the natural inducer for lac operon,
however, in the laboratory, an artificial inducer IPTG is always being used. Why?
3.
What is the importance of SDS in this experiment?
4.
Do you think the proteins will migrate from positive to negative pole or the reverse?

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Lab Manual 201505
Bachelor of Science (Hons) Microbiology