chrisb at hgu.mrc.ac.uk
Wed Jan 17 07:50:47 EST 1996
jlight at RESUNIX.RI.SICKKIDS.ON.CA wrote:
: Does anyone out there have experience doing BsaBI digests? It's one of
: those enzymes with activity that's blocked by overlapping dam methylation.
: I'm trying to cut a plasmid with a single BsaBI site. The plasmid was
: recovered from strain GM2163, which is dam(-) and dcm(-), but the digest
: doesn't appear to be working any better. Any advice would be greatly
I presume you're remembering to do the digests at 60 C, not 37 C.
You could check the enzyme out by restricting any eukaryotic
chromosomal DNA and looking for the characteristic smear on a gel. As
Duncan mentioned, you can check out the plasmid DNA from GM2163 with
MboI, which only cuts Dam- DNA. If like most people you don't have
MboI, TaqI might be useful -- it won't cut GATCGA or TCGATC sequences,
so (if your plasmid contains at least one of these), the TaqI digestion
pattern of Dam+ plasmid will differ from that of Dam- plasmid.
Someone else mentioned that you need to propagate GM2163 on
chloramphenicol to maintain the dam mutation (which is caused by a Tn9
insertion). I disagree. The frequency of spontaneous perfect excision
of Tn9 is negligibly low.
Chris Boyd | from, \MRC Human Genetics Unit / Western General Hospital
chrisb at hgu.mrc.ac.uk| not for \ Crewe Road / Edinburgh EH4 2XU / Scotland
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