HELP -DNA extraction from phage

Mayumi Yagi myagi at u.washington.edu
Thu Jul 27 11:22:21 EST 1995


Hi, Jacqueline,

One thing you might try is to resuspend the phage particles in STE and 
200ug/ml Proteinase K.  Incubate for an hour or so at 55, then do your 
phenol/chloroforms.  The EDTA will blow the phage particles apart, 
but also kills DNase, and the Proteinase K will digest both the phage 
proteins and any other nucleasesthat happen to be around.  By the way, 
are you adding DNase and RNase to your phage lysates before the PEG 
precipitation?  Adding them helps get rid of contaminating bacterial NA 
and cleans up your prep (otherwise all that stuff comes down with the 
PEG/NaCl).  We used to do this routinely and didn't have any problems 
with degradation if we used proteinase K in the later DNA prep.

Good luck, hope this helps.

On 26 Jul 1995, jacqueline heard wrote:

> Hi netsurfers and gene jocks,
> 
> 	I am in need of new insights and/or protocols to isolate DNA from
> bacteriophage.  
> I have used a phage kit from Bio 101 and my results were far from 
> exemplary.  The protocol I am currently using has allowed me to get
> excellent DNA
> yields but the purity is subpar, mucho degradation.  The degradation is
> preventing me from subcloning.  The procedure calls for
> precipitating the phage with PEG and NaCl,
> chilling for one hour, and then spinning at 10,000rpms for fifteen
> minutes.  After
> resuspending the pellets in SM, the protocol calls for phenol/chloroforms
> until the interphase is devoid of any residual phage proteins. From there
> I do an ethanol 
> precip. and wash.  I have minimalized the time that the DNA is in contact
> with the buffered  phenol by doing repeated one mintue aggitations and
> spins until the
> interphase is clean.  However, to no avail, this hasn't prevented my
> degradation.
> If anyone out there has any advice or new strategies, I would greatly
> appreciate
> hearing them. I'm back. I'm back.
> 
> Respectfully yours,
> 
> John Lesher
> 
> 
> 
> 
> 
> 
> 



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