ISOLATION OF PLASMID DNA FROM Escherichia coli:

By STET (Rapid) method.

Aim:
To isolate the plasmid DNA from Escherichia coli harboring pUC 18 plasmid by alkaline
lysis method.
Principle:
In the first step of the experiment a cell lysis solution is added to the cells. This solution
contains the detergent Triton X which dissolves the cell membrane and denatures proteins.
Sucrose helps to maintain osmotic regulation in and outside the cell. Under the presence of STET
buffer and enzyme lysozyme addition, the two strands in non-supercoiled DNA (linear fragments
of chromosomal DNA, relaxed and nicked circular DNA) separate and are partially removed
from solution.
However, this doesn’t occur with supercoiled forms of plasmid DNA because the two strands
are intertwined and entangled in a way that prevents them from coming apart. Therefore,
supercoiled plasmid remains free in solution. The chromosomal DNA of E. coli is attached at
several points to the cell membrane. Centrifugation also removes large amounts of entrapped
chromosomal DNA. Tris buffer (diluted buffer concentrate for RNase) is used to resuspend the
DNA precipitate in a higher concentration. The buffer contains the enzyme RNase, which further
degrades RNA. The concentrated gel loading solution prepares the sample for electrophoresis by
making it denser than the electrophoresis buffer. This enables the sample to sink into the wells of
the submerged gel. A negatively charged, blue tracking dye is also present to monitor the
electrophoresis and to make sample loading easier.
Materials:
Buffers and Solutions
(i)Antibiotics for plasmid selection (Ampicillin & Kanamycin)
(ii) STET Buffer
(iii) 5% cTAB
(iv) 1.2 M NaCl
(v) Lysozyme
STET Buffer
Sucrose 8g
Triton X 100 100 µl
1M Tris, pH 8.0 5 ml
EDTA (0.5M) 10 ml
Make up to 100 ml
Make sure that the pH of STET is 8.0 after all ingredients are added. There is no need to
sterilize STET before use.
Enzymes and Buffers
Lysozyme (10 mg/ml)
TE (pH 8.0) containing 20 µg/ml RNase A
EDTA (0.5M, pH 8.0)
To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA. 2H 2O to 800 ml of H2O.
Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH
pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not
go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.
Lysozyme (10 mg/ml)
Dissolve solid lysozyme at a concentration of 10 mg/ml in 10 mM Tris-Cl (pH 8.0) immediately
before use. Make sure that the pH of the Tris solution is 8.0 before dissolving the protein.
Lysozyme will not work efficiently if the pH of the solution is less than 8.0.
NaCl (1.2 M)
To prepare a 1.2 M solution: Dissolve 70.128 g of NaCl in 800 ml of H 2O. Adjust the volume to
1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at
room temperature.
CTAB (5%)
To prepare 5% CTAB (Hi Media) Solution: Dissolve 5 g of Cetyl TriMethyl Ammonium
Bromide in 100 ml of distilled water. Sterilize by autoclaving. Store the solution at room
temperature.

TE (10mM, pH 8.0)
100 mM Tris-Cl
10 mM EDTA (pH 8.0)
(10x Tris EDTA) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on
liquid cycle. Store the buffer at room temperature.
Procedure:
1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate
antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C
with vigorous shaking.
2. Take 1.5 ml of the culture into a microfuge tube.
3. Spin at 13,000 rpm for 20 seconds and Pour off the supernatant
4. Vortex the eppendorf to mix the pellet with remaining LB.
5. Resuspend the bacterial pellet in 200 µl of STET.
6. Add 5 µl of a freshly prepared solution of lysozyme. Close the top of the tube and mix the
contents by gently vortexing and incubate for 2 minutes.
7. Boil for 45 seconds and spin at 13,000 rpm for 10 min and remove pellet alone by toothpick.
8. Add 10 µl of RNase (1 mg/ml) and incubate at 68 0C for 15 minutes.
9. Add 20 µl of 5 % cTAB, spin for 5 min and decant the supernatant.
10. Resuspend in 300 µl of 1.2 M NaCl by vortexing
11. Add 300 µl of 100% ethanol, spin for 10 min at 13,000 rpm
12. Final rinsing with 500 µl of 70% ethanol, spin for 2 min, then decant and keep the pellet
13. Air dry and resuspend in 20 µl TE
Gel casting and sample loading procedure:
1. Seal the two ends of the gel casting tray using cellophane.
2. Prepare 30 ml of 0.8% agarose solution using TBE buffer.
3. Boil it till the agarose gets dissolved completely
4. Add 3 µl of Ethidium Bromide (Et-Br) to the agarose solution
5. Cool to a tolerable temperature (50-60 oC) and pour into the gel casting tray with the comb
placed. Allow it to solidify
6. Remove the comb and cellophane once the gel solidified.
7. Mount the gel in the electrophoretic tank and fill the tank with TBE buffer till the gel sinks
8. Mix 10 µl of DNA sample with 2 µl of Gel loading dye in a glass slide.
9. Load the samples into the wells and perform the electrophoresis at 50V.
Result and Discussion:

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