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RESEARCH DIVISION Laboratory Manual

 


 

Mouse and Human Genomic DNA Extraction

Treat human blood and supernatants as potentially infectious.  Discard supernatants by an appropriate method, eg into bromochlor solution.

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DNA extraction from frozen blood:

  1. Collect 10ml blood into a heparin or EDTA tube.
  2. Transfer to a 50ml falcon tube and freeze overnight at -20C.
  3. Allow to thaw in iced water for several hours.
  4. Add 40ml of triton/sucrose lysis buffer to blood and invert to mix. Spin samples at 3000rpm for 15 minutes. Discard supernatant.
  5. Add 20ml of triton/sucrose lysis buffer and resuspend white cell pellet with a blue tip. Spin samples at 3000rpm for 10 minutes. Discard supernatant.
  6. Add 3ml salting out/lysis buffer to the white cell pellet. Resuspend using a blue tip.   Add 200l 20% SDS and 300l 5mg/ml proteinase K. Incubate samples overnight at 50C in a gently shaking incubator. If sample has not been completely digested   overnight, add 50l 5mg/ml proteinase K and continue incubation for several more hours.
  7. Add 1.5ml 6M NaCl and shake vigorously for 15 seconds. Spin sample at 3000rpm for 15 minutes.
  8. Transfer supernatant to a new tube, and add 10ml absolute ethanol. Allow the DNA to come out of solution. Hook out DNA and transfer to an ependorf tube. Spin briefly and remove ethanol. Add 400l TE and allow DNA to resuspend overnight at 4C.
  9. Extract once with phenol/chloroform and once with chloroform. Ethanol precipitate and rinse pellet with 70% ethanol. Allow pellet to resuspend overnight at 4C.

Triton/sucrose lysis buffer1 litre:     

1M MgCl2 5ml
1M Tris-Cl pH 7.5 10ml
Triton X-100 10ml
sucrose 109.54g

Salting out/lysis buffer100mls:

1M Tris-Cl pH 7.5 1ml
5M NaCl 8mls
500mM EDTA pH 8 400l

 

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Mouse:

  1. Dissect one lobe of liver tissue, and freeze in liquid nitrogen.
  2. Grind tissue into a fine powder under liquid nitrogen.
  3. Transfer tissue to a cold 15 ml blue top tube, add 5 ml of salting out/lysis buffer (10 mM Tris-Cl pH 7.5, 400 mM NaCl, 2 mM EDTA pH 8) and resuspend tissue with a blue tip.
  4. Add 200 l 20% SDS and 300 l 5 mg/ml proteinase K. Incubate samples for
  5. several hours at 50C, until tissue had been completely digested.

 

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Extraction and precipitation:

  1. Add 5 ml phenol equilibrated with 500 mM Tris pH 8, place on slowly rotating wheel for 15 minutes. Centrifuge 3500 rpm for 10 minutes. Remove aqueous phase to another tube.
  2. Add 5 ml phenol/chloroform equilibrated with 500 mM Tris pH 8, place on slowly rotating wheel for 15 minutes. Centrifuge 3500 rpm for 10 minutes. Remove aqueous phase to another tube.
  3. Add 5 ml chloroform equilibrated with TE, place on slowly rotating wheel for 15 minutes. Centrifuge 3500 rpm for 10 minutes. Remove aqueous phase to another tube.
  4. Add 0.5 vol isopropanol and allow DNA to come out of solution. Gently swirl the tube for a few minutes. (**At this stage, tube can be placed at -20C overnight.)
  5. Hook out the DNA with a pasteur pipette hook, and allow excess solution to drain from the DNA by holding the blob to the side of the tube.
  6. Place hook with DNA on it into a 15 ml blue top tube with 1 ml TE. Break off pasteur pipette and leave hook with DNA in tube. Place on very gently rocking platform for several hours until DNA has completely gone into solution.
  7. Quantitate and resuspend DNA at a final concentration of 500 g./ml.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998