Isolation Of Plasmid DNA By Using Alkaline Lysis

Plasmid is an extra chromosomal, circular, self-replicating double stranded DNA molecule found in bacterial cells. Many bacteria and few eukaryotes contain these elements. Plasmids confer resistance to antibiotics, heavy metals, sensitivity to mutagens, resistance to bacteriophages, restriction enzyme production, rare amino acids and catabolism of complex organic compounds. Some of them freely transfer into other bacterial species and others have transferred their plasmid DNA into E. coli.

Many artificial plasmids have been constructed in vitro by using fragments of DNA from these naturally occurring plasmids. Some are frequently used as replicator, marker and a site for cloning.

Requirements

  • LB medium*
  • TE buffer (pH8)
  • NaOH/SDS solution
  • Potassium acetate solution
  • Ethanol (70% and 100%)
  • DNase free RNase (10 mg)

*LB (Luria Brutini) medium:

  • Bactotryptone 10 g
  • Yeast extract 5 g
  • NaCl 10 g
  • Distilled water 1 litre
  • Agar 15 g (if solid medium is to be prepared)

Procedure

  1. Inoculate 10 ml LB medium with a single bacterial colony and incubate to grow overnight.
  2. Centrifuge the bacterial growth in microcenrifuge, bacterial pellet is formed.
  3. Suspend pellet in 100 ml TE buffer for 5 minutes.
  4. Add 200 mg/ml NaOH/SDS solution, tap it with finger to mix properly.
  5. Keep it on ice for 5 minutes, centrifuge the tube for 1 min to form pellet. The supernatant contains plasmid.
  6. Transfer it into other fresh microcentrifuge and add 0.9 ml 100% ethanol into it
  7. Remove supernatant and wash pellet with 1 ml of 70% ethanol and dry under vacuum evaporator.
  8. Further suspend it in 20 ml TE buffer.
  9. Now it is ready to be used for restriction digest.
  10. Take the sample to gel electrophoresis, allow the DNA fragments to move under the influence of electric current as per instruction mentioned on the kit.

Results

Different bands will be visible on agarose gel at various distance based on the size of DNA fragments. The small sized DNA will be on the to and the small sized below to them.

Isolation of plasmid DNA

Reference:

Practical Microbiology

Gaurav Singh

Editor in Chief Medical Microbiology & RDT Labs - RDT Labs Magazine

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