HELP -DNA extraction from phage

[Martin Osterhoff]

Martin Osterhoff <Martin.Osterhoff at rz.ruhr-uni-bochum.de

Hi Jacqueline,

before all try to avoid too much agitating the DNA. I only turn around
the tube for several times when extracting or tip against it with a
finger.
Make two or three chloroform extraction after PEG precipitation and
centrifugation. It is really necessary to get rid of PEG rests which
otherwise will bind to DNA afterwards.

I wish you good results,

                                      Martin



jacqueline heard <jheard at hermes.bc.edu> 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

>

>
>