Restriction Digestion of Phage DNA

Wallace Lauzon wlauzon at uottawa.ca
Fri Sep 25 14:36:57 EST 1998


Hi,

Just a shot in the dark.  Are the DNA samples relatively dilute and
resuspended in TE buffer after precipitation?  The EDTA may inhibit the
reactions if it makes up a large enough component of the digestion
reaction.

Hope it helps,

Wallace Lauzon



Jay Mone' wrote:
> 
> As a laboratory experiment, I and some students have isolated several
> E. coli specific bacteriophages from untreated sewage.  We have
> isolated the phage DNA by treatment of purified phage preps by
> treatment with proteinase K and SDS, followed by phenol-chloroform
> extraction and precipitation with alcohol.  The problem is that all of
> the phage DNA preps appear to be completely resistant to restriction
> digestion.  I have tried several enzymes, including EcoRI, HindIII,
> PstI and KpnI, and never a hint of digestion?  What is going in here?
> I generally run lambda DNA as a control to ensure that the enzymes
> cut, so thats not the problem.  The 260/280 is consistently > 1.5.
> Help!!
> 
> Jay Mone''




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