DNA isolation from Rhizobacteria

Written by Super User. Posted in Botany

Learn about DNA isolation from rhizobacteria.

rhizobacteria

 

Alok Kumar Varshney, M. KrishnaMohan and P. Ghosh.
Birla Institute of Scientific Research, Statue circle, Jaipur (Rajasthan) India

Reagents for DNA Isolation from Rhizobacteria

  • TE Buffer (10mM Tris and 1 mM EDTA, pH-8.0).
  • 10% SDS
  • ProteinaseK (20mg/ml)
  • 5M NaCl
  • 10% CTAB in 4% NaCl
  • Chloroform:Isoamylalcohol (24:1)
  • 100% Ethanol
  • 70% Ethanol

Procedure for DNA Extraction

  1. Take 245μl TE buffer in eppendorf tube and add one loopful bacterial growth from fresh plate.
  2. Give freeze-thawing treatment by placing the tube in boiling water for 5 minutes and then snap chilled in ice at -20oC for 20 minutes. This makes the cell wall ruptured.
  3. Add 25μl SDS (10%) and 2.0μl ProteinaseK (20mg/ml) and incubate for 30 minutes at 65oC in shaking water bath.
  4. Now add 40μl of 5M NaCl and 30μl of CTAB-NaCl, mix gently and incubate for 30 minutes at 65oC in shaking water bath.
  5. Add 342μl Chloroform:Isoamylalcohol (24:1) and vortex gently.
  6. Spin at 10000 rpm for 15 minutes.
  7. Collect aqueous phase in a new tube and add 1 volume 5M NaCl and 2 volume ice chilled absolute ethanol and incubate for 30 minutes at -20oC.
  8. Spin at 10000 rpm for 15 minutes.
  9. Discard the supernatant carefully and add chilled 70% ethanol.
  10. Again spin at 10000 rpm for 15 minutes.
  11. Discard the supernatant carefully without disturbing the pellet.
  12. Dry the tube at room temperature for removing any trace of ethanol.
  13. Resuspend the pellet in 50μl TE buffer.

Submitted by : Alok Kumar Varshney.

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