pCaSpeR-hs cloning problem
6kuritaro at earthlink.net
Sun Apr 6 21:12:26 EST 2003
Hi there --
I have a student trying to subclone a 4.1-kb Xba fragment into
pCaSpeR-hs. The initial subcloning, from a genomic BAC into NEB's
pLITMUS went without a hitch, but she's not been able to transfer the
insert into pCaSpeR-hs, which is about 8.8 kb.
Cut both with Xba, gel-purify, dephosphorylate the vector*, ligate,
transform competent JM109. Analyze plasmids from transformants by
cutting with Xba and we see two constructs:
a single 8.8 kb fragment which is presumably recircularized vector
a 4.1 kb fragment plus a roughly 2.7 kb fragment -- I'm guessing the 4.1
is our insert (though I haven't tested that yet), but what the heck is
up with the vector?
(* we get the same two plasmids back if we don't dephosphorylate the
vector, so that doesn't seem to be the problem.)
We've done this three times now, always with the same results. I'm going
to spend a little time trying to troubleshoot, but I'm hoping someone
reading the newsgroup has heard of this, or better still has had some
experience of it. Thanks for any suggestions,
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