In article <360BA6A2.77FE at marauder.millersv.edu>, Jay Mone'
<jmone at MARAUDER.MILLERSV.EDU> 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''
Ah, how do you know your phages produce double stranded DNA like lambda
and not single stranded like M13 or single stranded RNA like MS2.
Spike your sample DNA with lambda and digest with say Hae III. This is a
4 cutter so should chew the lambda up nicely and you should also have
some sites in a double stranded DNA phage. I would avoid hexameric RE's
for now. If the lambda cuts then you have no inhibitors in your samples
and if the sample also cuts then it is double stranded and probably had
no sites for the enzymes you originally tried. If the lambda cuts and
your sample doesn't then I would suspect the sample is not double
stranded. In this case a plasmid prep of the E.coli should show the
double stranded RF form of the phage. There are other checks. Do you get
turbid plaques or total lysis. You could also be picking up T-even like
phages which modify their DNA such that 99.9% of RE's won't cut it.
Interesting to find out what you really have there. I'm sure you can
work out further ways to test for ssDNA and RNA phages etc.
What E.coli did you use for isolation. If it was F+ then you will pick
up the M13 like phages.
Good luck.
Duncan
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Duncan Clark
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