In article <360BA6A2.77FE at marauder.millersv.edu>
jmone at MARAUDER.MILLERSV.EDU ("Jay Mone'") writes:
> 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''
Most likely you have dirty dna - some contaminant perhaps even a little
phenol, which is inhibiting digest. One quick way to see if you have a
contaminant, is to mix your lambda DNA with your sample DNA and see if
the lambda still cuts.
Alternately, some coliphages use modified nucleotides and therefore
don't cut with restriction enzymes. T4 for example.
__________________________________________________________
Michael Benedik
Department of Biology and Biochemistry
University of Houston Tel: 713-743-8377
3201 Cullen Boulevard, HSC 402 Fax: 713-743-8351
Houston, TX 77204-5934 email: benedik at uh.edu
__________________________________________________________
