P1 Purification Help Needed!
smori at nmsu.edu
Sun Nov 26 18:42:29 EST 1995
Karina McQueen (mcqueen at unixg.ubc.ca) wrote:
: Hi netters -
: I am looking for a fast and easy way to purify DNA from P1
: bacteriophage clones. I have at least 15 samples to do so a fast,
: efficient and easy approach would be ideal. I have been trying
: CsCl preps, but have had minimal success! Any information
: regarding special reagents or columns would be appreciated.
I don't quite understand your problem. I am guessing you have some liquid
culture of E coli (?) infected with P1. Now you have grown this stuff
and wanna isolate the DNA in the phage that's present in the sup??
We add solid PEG to the sup after pelleting the cells in the cfg. Add PEG
to the final 5% w/v. Then add solid NaCl to final  of 0.5M.
dissolve and leave on ice for 30min with occasional mixing.
Spin at max speed in micro cfg for 15min -> drain sup and spin for
another 10min. take out the remaining liquid. Resuspend in desired TE buffer.
Do Phenol/Chloroform Extractions 3 times; followed by 2X Chloroform
extraction. Back Extract with TE (*pretty important* at end of each
Add 1/10th vol 3M NaOAc and 3X vol Cold 95% ETOH and ppt at -70C for
30-60min. You can add 5ul of 10mg/ml Glycogen to increase the pptation....
Voila -> Phage DNA to feast on.
C A N A D A
Program in Molecular Biology
Department of Chemistry and Biochemistry Box 3C
New Mexico State University
Las Cruces NM
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