Isolation of plasmid DNA by boiling lysis method and analysis by gel electrophoresis

/Isolation of plasmid DNA by boiling lysis method and analysis by gel electrophoresis
Isolation of plasmid DNA by boiling lysis method and analysis by gel electrophoresis 2018-02-23T12:24:14+00:00

Isolation of plasmid DNA by boiling lysis method and analysis by gel electrophoresis

Isolation of plasmid DNA by boiling lysis method and analysis by gel electrophoresis

Introduction

The bacterial cell is enclosed in a cytoplasmic memberane and surrounded by a rigid cell wall with same species including E.coli the cell wall may itself be enveloped by a second outer memberane. All of these barriers have to be disrupted to release the cell components. The chemical used to break the cell wall are sodium chloride, Tris, EDTA and Triton X- 100 (STET). Tris is used to maintain the pH, EDTA removes magnesium ions that are essential for preserving the overall structure of the cell envelope. Triton X – 100 helps in the weakning of cell wall. Lysozyme is also used in this method. It is an enzyme that is present in egg, tears and saliva. Cell are then lysed by heating to 100ºC. In addition to cracking, heating disrupts base pairs of DNA strands and denatures the proteins and chromosomal DNA of the host. However, the strands of closed circular plasmid DNA are unable to separate from each other because their phosphodiester backbone are topologically interwined, when the temperature is lowered the bases of closed circular molecules of DNA falls once again into register forming super helical molecules. After denatured, chromosomal DNA and proteins have been removed by centrifugation, native plasmid DNA can be recovered from the supernatant.

Requirements

Reagents

  1. LB media
  2. MCT
  3. Tris Cl (10mM)
  4. EDTA (1mM)
  5. 0.1M NaCl
  6. Triton X 100
  7. Lysozyme (20mg/ml)
  8. 3M NaAc
  9. Isopropanol
  10. 70% ethanol

Equipments

  1. Water bath
  2. Distilled water

Procedure

  1. Pellet down the overnight grown culture at 8,000 rpm at RT for 5minutes.
  2. Remove the supernatant and to resuspend the pellet add 600µl of STET to it.
  3. Vortex the MCT and add 20µl of lysozyme. Invert the MCT to mix the contents and keep it at room temperature for 10minutes.
  4. Boil the MCT at 100ºC for 2minutes.
  5. Spin the MCT at 12,000rpm for 15minutes at RT.
  6. To the supernatant add 200µl of 3M NaAc and 400µl of isopropanol to it.
  7. Centrifuge the tube at 12,000rpm at RT for 10minutes.
  8. Discard the supernatant and to the pellet add 200µl of 70% ethanol.
  9. Again centrifuge the tube at 12,000 rpm at RT for 5minutes.
  10. Dry the ethanol from the MCT in the incubator.
  11. Dissolve the pellet in 40µl of autoclaved distilled water.
  12. Prepare the reaction mixture and load it on the gel.
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