Home | Jobs | News & Articles | Job Advice | Search | Protocols | Fun | @RealLabRat
Lab Rat with DNA

Post your resume
Candidates - post your resume

Search job listings
Candidates - search biotech jobs

Post job openings
Employers - post job openings

HOME > Protocols > Bacterial > Bacterial DNA > Protocol for Preparation of Genomic DNA from Bacteria

Protocol for Preparation of Genomic DNA from Bacteria
(Modified from Experimental Techniques in Bacterial Genetics, Jones and Bartlet, 1990)

  1. Transfer 1.5 ml media to a micro centrifuge tube and spin 2 min. Decant the supernatant
  2. Drain well onto a Kimwipe
  3. Resuspend the pellet in 467 l TE buffer by repeated pipetting
  4. Add 30 l of 10% SDS and 3 l of 20 mg/ml proteinase K, mix , and incubate 1 hr at 37oC
  5. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
  6. Carefully transfer the DNA/phenol mixture into a Phase Lock Gel tube and spin 2 min.
  7. Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform.
  8. Again mix well and transfer to a new Phase Lock Gel tube and spin 2 min.
  9. Transfer the upper aqueous phase to a new tube.
  10. Add 1/10 volume of sodium acetate.
  11. Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates.
  12. Centrifuge to pellet DNA.
  13. Wash in 70% Ethanol.
  14. Resuspend DNA in 100-200 l TE buffer.
  15. After DNA has dissolved, measure the concentration by diluting 10 l of DNA into 1 ml of TE (1:100 dilution) and measure absorbance at 260 nm.
  16. Concentration of original DNA solution in g/ml = Abs x 100 x 50 g/ml.

Caution: Phenol causes severe burns! Wear gloves, goggles and lab coat! Keep tubes capped tightly.


theLabRat.com 2005. All Rights Reserved.