Biology Protocols

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PEG Purification of Plasmids

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Carry out a normal plasmid prep to the phenolization step. Phenolize two or three times, chloroform once and ethanol precipitate. Spin down, wash and drain thoroughly. Dry the pellet fairly well, but complete dryness does not seem to be essential.

  • Resuspend in 1.68ml of water and then add:

                                              0.32ml 5M NaCl
                                              2ml of 13% PEG 8000

  • Mix thoroughly and leave on ice and water for more than 1h (overnight is possible).
  • Spin down (10K; 20m; 4°C), wash, dry, resuspend in 250µl of water.
  • Take samples.
  • If the DNA seems free of RNA, make the solution 0.3M with NaCl, CHCl3 x 5 and etherize once with a 150µl 0.3 TNE followthrough.
  • Ethanol precipitate, wash, dry and resuspend in 250µl of water.

Notes

If the DNA is very big, be prepared to dilute at stage 5, the solution can become very viscous if all of the PEG is not removed.

 


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