P1 DNA PEG precipitation

P1 DNA PEG precipitation

bionet.molbio.methds-reagnts Nov 26 1995

With liquid culture of E coli infected with P1.
  1. add solid PEG to the supernatant after pelleting the cells. Add PEG to the final 5% w/v.

  2. Then add solid NaCl to final [] of 0.5M. Dissolve and leave on ice for 30min with occasional mixing.

  3. Spin at max speed in micro cfg for 15min -> drain sup

  4. spin for another 10min. to take out the remaining liquid.

  5. Resuspend in desired TE buffer.

  6. Do Phenol/Chloroform Extractions 3 times

  7. followed by 2X Chloroform extraction. Back Extract with TE (*pretty important* at end of each Chloroform step).

  8. Add 1/10th vol 3M NaOAc and 3X vol Cold 95% ETOH

  9. ppt at -70C for 30-60min. You can add 5ul of 10mg/ml Glycogen to increase the pptation....

  10. Voila -> Phage DNA to feast on.

smori@nmsu.edu (Shahram Mori)