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Isolation of plasmid DNA from Escherichia coli.

1. Objective:

Isolation of plasmid DNA from Escherichia coli.

2. Introduction

Plasmids are the circular, autonomously replicating, extra-chromosomal double-stranded DNA molecules found in bacteria, cyanobacteria, fungi, etc.

They are classified according to the phenotypes conferred by them on the host cells e.g. antibiotic resistance, fertility (F) factor, etc. Naturally occurring plasmids also encode for genes that are required for conjugation e.g. F-plasmid.

There are three forms of plasmids: super-coiled, relaxed and linear. In recombinant DNA technology, plasmids are used as cloning vehicle or vector to introduce foreign genes into a host organism.

There are several methods given for plasmid isolation, but the most commonly used method is called ‘alkaline lysis’.

3. Principle :

In alkaline lysis method, the cells are lysed using EDTA (that chelates metal ions) and a SDS detergent. It weakens the bacterial cell wall and also inactivates the enzymes digesting the DNA (DNases). SDS removes lipid molecules, disrupts the cell membrane and also denatures the bacterial proteins.

After adding NaOH, pH of the solution increases to 11-12. Hence, it denatures the bacterial chromosomal DNA and the plasmid DNA. When the pH is reduced after adding potassium or sodium acetate in the solution, the plasmid DNA renatures because of its small size.

But the chromosomal DNA strand and bacterial proteins form a precipitate along with SDS. You can remove the precipitate by centrifugation and adding isopropanol and can concentrate the renatured plasmid in solution. You can remove the contaminating RNA by digesting through RNase.

4. Requirements :

i. Tris, sodium dodecyl sulphate (SDS), tryptone, yeast extract, sodium chloride, agar, potassium acetate

ii. Boric acid, glucose, EDTA, sodium hydroxide

iii. Isopropanol, distilled water, high speed refrigerated centrifuge, pH meter micropipettes, water bath, vortex mixer

iv. Agarose gel electrophoresis apparatus, Eppendorf tubes, Tips, glassware

Preparation of Reagents:

The following solutions should be prepared in advance:

(i) 1M glucose, 1M Tris (pH 8.0), 0.5 MEDTA (pH 8), 5 N sodium hydroxide (autoclave these solution separately and store at room temperature)

(ii) 10% SDS: Store at room temperature

(iii) Ampicillin solution: Dissolve 100 mg penicillin in 1 ml sterile distilled water to get stock solution of 100 mg/ml; store at -20°C or 4°C. The concentration of ampicillin in any medium is 50-100 pg/ml. Do not autoclave this solution.

(iv) Luria Bertani (LB Medium): Add 1 g bactotryptone, 0.5 g yeast extract, 1 g sodium chloride; adjust the pW to 7 with 1 N NaOH and make up the volume to 100 ml with water. Autoclave the medium and store at room temperature.

(v) Solution I: Add 5 ml of 1M glucose (50 mM final concentration of glucose), 0.25 ml of 1M Tris (pH 8) (25 mM final concentration of Tris, pH 8) and 2 ml of 0.5 MEDTA (pH 8.0) (lOmM final concentration of EDTA). By using water make up the volume to 100 ml Autoclave the solution and store at room temperature.

(vi) Solution II: Prepare fresh solution on the day of the experiment by mixing 4 ml of 5N NaOH (0.2 N final concentration of NaOH) and 10 ml of 10% SDS (final concentration of 1% SDS). By using autoclaved water make the volume of solution to 100 ml. Discard the solution after use.

(vii) Solution III: Prepare 3 M CH₃COOK solution of pH 4.8. Autoclave it and store at 4°C.

(viii) Running Buffer (for electrophoresis): There are two common types of running buffers used in agarose gel electrophoresis : Tris-Borate-EDTA (TBE) and Tris-Acetate-EDTA (TAE). Prepare the following stock solutions:

5X TBE, 1 Litre: 54 g Tris, 27.5 g boric acid, 20 ml 0.5 M EDTA, /?H8.0

50X TAE, 1 litre: 24.2 g Tris, 5.71 ml of glacial acetic acid, 20 ml of 0.5 M EDTA, pH8.0.

(ix) TE: Add 1 ml of 1M Tris, pH 8 (10 mM Tris, pU 8.0) and 0.2 ml of 0.5 M EDTA, pU 8.0. (ImM final concentration of EDTA). Using distilled water make the volume to 100 ml. Autoclave the solution and store at room temperature.

(x) 6X Gel-Loading Buffer: 0.15% bromo-phenol blue, 0.15% xylene cyanol, 30% (v/v) glycerol in water.

(xi) Ethidium Bromide Solution: Dissolve 10 mg/ml in water. Store at 4°C wrapped in an aluminium foil (wear gloves while working with this dye because it is mutagenic).

(xii) IX-TBE, ILitre: Dilute with water 200 ml 5X stock of TBE so as to get 1 liter.

Inoculate a single bacterial colony into 2 ml of autoclaved LB medium containing 2 pi of 100 mg/ml ampicillin solution. Incubate the culture at 37°C overnight with shaking (200-250 rpm) condition.

5. Procedure :

(i) After 24 hours incubation, take 1.5 ml culture from the 2 ml culture using an eppendorf tube pipette.

(ii) Centrifuge the cells at 6,000 rpm for 5-10 minutes. Discard the supernatant completely by inverting the eppendorf tube on the blotting paper. Put the eppendorf tube on ice.

(iii) Completely re-suspend the pellet in (0.1 ml) of ice cold Solution I to get a uniform suspension. Put on ice for 5 minutes then keep at room temperature.

(iv) To this suspension, add 0.2 ml of freshly-prepared Solution II. Tube should be closed tightly. Properly mix the contents by inverting the tube five times.

(v) Add 0.15 ml of ice-cold Solution III. Close the tube tightly and mix the contents properly by inverting the tube. Keep for 5-7 minutes on ice. Vortex at 10,000 to 12,000 rpm for 10 minutes at 4°C.

(vi) Soon transfer the supernatant to a fresh eppendorf tube and add 0.45 ml of isopropanol. Gently mix by inverting the tube and keep at room temperature for 10 minutes. Centrifuge at 10,000 rpm for 20 minutes at room temperature. Take out the supernatant.

(vii) Thereafter, add 0.1ml of 70% ethanol into the pellet and spin at 10,000 rpm for 5 minutes at 4°C. Then discard the supernatant carefully and dry the tube at 37°C so that any traces of isopropanol could be removed.

(viii) Now, add 20 (il of IX-TE from the side and gently tap the tube with your fingers. Add 3.33 (il of 6X gel loading buffer and run on 1% agarose gel as it has been described below (Note: alternatively, a plasmid-isolation kit of any company can be used).

Preparation of 1% agarose gel and set up of electrophoresis:

It follows the following steps:

(i) Dilute 50X TAE or 5X TBE buffer with distilled water to get IX TAE or IX TBE.

(ii) Pour 50 mL of IX TAE or IX, TBE buffer in a 250 ml conical flask and add 0.5 g of agarose into it. Boil to get a clear solution and cool to warm a liquid (60°C).

(iii) In the electrophoresis set put the combs in such a way that it should be about 2 cm away from the cathode.

(iv) Add ethidium bromide (10 mg/ml stock) to make the a final concentration of 0.5 |ig/ml of gel when the temperature of agarose gel is around 60°C.

(v) Gently pour the solution into the gel tank. Pour the agarose gel in such a way that it could be 0.5 – 0.9 cm thick and without air bubbles. Allow the gel to get solidified.

(v0 Load the samples into the well very carefully and record that which sample is being loaded into which well as Lane 1, Lane 2, and so on. Start the power connection and set voltage to 50 V.

(vii) Until the second dye (blue dye) has reached % of the gel, run the gel. (it takes about 1 hour). Observe under UV trans- illuminator.

6. Result :

After precipitating with isopropanol and centrifugation, a white precipitate is observed on the sides or at the bottom of the centrifuge tube. Usually 2 bands are observed when the plasmed DNA is run on an agarose gel.

These are the supercoiled and relaxed or open circular form of the plasmid. During plasmid isolation no RNase was added; hence a band of RNA will also be seen on the gel. RNA moves faster than the DNA being small in size; therefore, RNA can be distinguished from the plasmid DNA.