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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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Lab Manual 201505
Bachelor of Science (Hons) Microbiology
Reference :
1. Sambrook, J., Russell, D. W., Sambrook, J. (2001). Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory
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Lab Manual 201505
Bachelor of Science (Hons) Microbiology
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)
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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Lab Manual 201505
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Time
Number of cycles
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Lab Manual 201505
Bachelor of Science (Hons) Microbiology
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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Lab Manual 201505
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Lab Manual 201505
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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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Microfuge tubes
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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Lab Manual 201505
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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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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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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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Lab Manual 201505
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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.
Equipment
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Lab Manual 201505
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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)
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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Lab Manual 201505
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B.
SDS-PAGE
Materials and Reagents:
Acrylamide/Bis
SDS
SDS-PAGE electrophoresis
apparatus
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
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Lab Manual 201505
Bachelor of Science (Hons) Microbiology