removal of PEG from DNA disolved in TE?

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Tue Feb 25 09:58:06 EST 1997


I would love to get your protocol, because I am having the same
problem.  I think the problem, im my case, results from the chloroform
in the phage lysate.  After the addition of the peg, the residual
chloroform dissolved in the phage lysate, causes some of the PEG to
ppt.  We got the same milky white prep.  I'm pretty sure that its PEG
because I phenol chloroform extracted it about five times and still
get a white interface each time.  There is less PEG now, but the
problem is not completely solved.  The intended use of the phage
library DNA is as a template for PCR, and I'm not sure how the PEG
will affect it.  Anyone have any experience?  Perhaps CTAB/NaCl and
chloroform extraction will be better at getting rid of the PEG.  Not
sure, but I'll probably try that next (barring any major revalations
from this newsgroup).

						Regards		SVEN


In article <Fncu8PAi$yCzEwmO at genesys.demon.co.uk>, "Dr. Duncan Clark" <duncan at genesys.demon.co.uk> writes:
> In article <vnp-1902971354020001 at mje-mac5.welc.cam.ac.uk>, Venkat
> Pisupati <vnp at mole.bio.cam.ac.uk> writes
>>Can anyone tell me how to remove PEG from my DNA solutions?
>>
>>I have lambda preps that I wish to cut and then spin in a microcon 100 to
>>separate the oligo linkers, but nothing goes through the filter because of
>>all PEG, I think  (the stock DNA solution is milky white and there is only
>>5-10 ugs of DNA in the restriction reactions that I am trying to spin.).
>>Chloroform extraction does not seem to do anything.
>>
> 
> Ethanol ppt and wash the DNA pellet with 70% ethanol. I have my doubts
> that your milky solution is PEG because PEG in solution up to at least
> 40% is clear. It would only be cloudy if it is ppting your DNA in that
> stock solution. I PEG ppt litres of phage at a time to prepare 100's of
> mgs of lambda and have never had a problem with PEG in the final DNA
> prep.
> 
> Duncan  
> --------------------------------------------------------------------------------
> The problem with being on the cutting edge is that you occasionally get 
> sliced from time to time....
> 
> Duncan Clark
> DNAmp Ltd.
> TEl/FAX 01252376288
> http://www.dnamp.com
> http://www.genesys.demon.co.uk



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