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There are quick and easy method to isolate Plasmid from Bacteria here I have elaborated the protocol for isolation of Plasmid from different type of bacteria.

1. Boiling method for small plasmids in E. coli
This method is simple and developed by Holmes & Quigley in1981 and modified by Riggs & McLachlan, 1986.

• For the isolation of plasmid first centrifuge 1.5 ml of culture in micro centrifuge tube and re-suspend pellet in 200 μl of STET buffer (8.0% sucrose, 0.5% Triton X-100, 0.05 M EDTA, 0.05 M Tris-HCl, pH 8.0) containing 10 μl of lysozyme (20 mg/ml, freshly dissolved in H2O) and 20 μl ZnCl2 (1% in H2O).

• Incubate at about 100°C for 50-55 sec, and then cool on ice

• Centrifuge for 5-10 min and add supernatant to micro centrifuge tube containing 480 μl of IS mix (400 μl isopropanol, 80 μl 5 M ammonium acetate). Incubate at RT for 20-30 min.

• Centrifuge for 5 min, wash DNA pellet with 70% cold ethanol twice and dry in a vacuum chamber.

• Re-suspend pellet in 20 μl of storage TE buffer or in RNase buffer before using for DNA gel electrophoresis.

2. Hot alkaline method for all plasmid sizes and bacteria

This method was developed by Kado & Liu in1981

• For the plasmid isolation by this method, Centrifuge 2-3 ml of culture in micro centrifuge tube, re-suspend pellet in 1 ml of solution containing 0.04 M Tris-acetate, pH 8.0 (adjust pH with glacial acetic acid) and 2 mM EDTA.

• Add 2 ml of lysis buffer (0.05 M Tris, 3% SDS, pH 12.50, adjusted with 2 N NaOH) and mix well.

• Incubate at ~65°C for 30-45 min (strain dependent)

• Add to hot samples 6.0 ml of phenol/chloroform (1:1) and mix gently to complete emulsification.

• Separate phases by centrifugation at 10,000 RPM for 10-15 min at room temperature and transfer the upper aqueous phase carefully (avoid inter-phase which contains debris) to new tube containing 1.0 volume of chloroform. Mix and centrifuge again for separation of phases.

• Recover aqueous phase and use directly for DNA gel electrophoresis.

3. Lysozyme method for various Gram-negative bacteria

This method is simple and developed by Davis et al.,1980

• For the plasmid isolation by this method, centrifuge 10 ml of culture, re-suspend pellet in 1.4 ml of TE buffer (0.01 M Tris, pH 8.5 and 1 mM EDTA). Transfer to micro centrifuge tubes and spin for 3 min.

• Re-suspend pellet in 0.4 ml of solution (15% sucrose, 0.05 M Tris, pH 8.5, 0.05 M EDTA), mix vigorously and cool on ice.

• Add 0.1 ml of freshly prepared lysozyme (5 mg/ml in TE buffer used above), mix carefully and incubate on ice for 20-40 min.

• Add 0.3 ml of pre-cooled Triton buffer (0.1% Triton X-100, 0.05 M Tris, pH 8.5, 0.05 M EDTA), incubate on ice for 20 min and centrifuge at 4°C for 4 min.

• Transfer clear supernatant into new tube and add 4 μl of di-ethyloxydiformiate, mix gently.

• Incubate for 15 min at 70°C, cool for 15 min to RT, then incubate on ice for 15 min.

• Centrifuge it for 4 min, transfer supernatant into new tube, fill up with -20°C ethanol for DNA precipitation and mix gently.

• Centrifuge for at least 30 min at RT, dry the pellet in vacuum and re-suspend in storage TE buffer or in RNase buffer.


4. Lysis of cells from single colonies

This method was developed by Eckhardt, 1978 and Priefer, 1984

• For plasmid DNA isolation by this method, transfer 1-2 freshly grown single colonies with a toothpick into 20 μl of cold buffer (0.025 M Tris, pH 8.0, 25% sucrose, 0.250 M EDTA, 7% Ficoll 400).

• Add 20 μl of freshly prepared lysis solution (0.1 mg/ml of lysozyme, 10 μl/ml of RNase A, in the above buffer), mix well and immediately fill with 10-15 of the mixture into the well of an agarose gel which contains 0.5% SDS

• Add as “upper layer” onto the cell lysate 10 μl of the following solution: 0.025 M Tris, pH8.0, 10% SDS, 25% sucrose, 0.07% bromophenol blue

• After 15-30 min apply low voltage (half of usual voltage) for 30 min, then apply usual electrophoretical conditions

5. Plasmid isolation from Gram-positive bacteria with mutanolysin or lysozyme
This method was developed by Klaenhammer in 1984

• For the isolation of plasmid first centrifuge 4 ml of culture and re-suspend pellet in 10 ml of fresh culture medium and Incubate for 1-2 hrs at 37°C

• Centrifuge again and re-suspend pellet in 1 ml of cold 25% sucrose, 0.05 M Tris, pH 7.5, 5 mM EDTA at 4°C.

• Keep cell suspension in ice bath for 10 min, then add 75 μl of either mutanolysin or lysozyme (1 mg/ml in 0.05 M Tris, pH 7.5, 5 mM EDTA), mix and incubate in ice bath for 1hr (for some strains incubation at 37°C for 1hr is preferred)

• Centrifuge cells and add 500 μl of the following lysis solution to the pellet and mix well (0.05 M Tris, 5 mM EDTA, 0.05 M glucose, 3% SDS) immediately before use mix 1.0 ml of this solution with 10 μl of 10 N NaOH.

• Heat the sample at 62°C for 1 hr, then allow to cool slowly (approx.15 min) to RT, add 50 μl of 2 M Tris, pH 7.0, mix gently and add 70 μl of 5 M NaCl and mix gently.

• Transfer into micro centrifuge tube and extract with 500 μl of phenol which is saturated with 3% NaCl (mix gently until emulsification), leave at RT for 5-10 min. Add 300 μl of chloroform and mix gently.

• Centrifuge for 5 min at RT for phase separation and take upper phase for extraction with 600 μl of chloroform:isoamylalcohol which is (24:1), leave at RT for 5-10 min, centrifuge and harvest aqueous phase for ethanol precipitation as usual.