PCR cloning into T-vectors

mcgraw%gandal.dnet at SERVER.UGA.EDU mcgraw%gandal.dnet at SERVER.UGA.EDU
Wed Feb 19 09:45:07 EST 1992


Bill,

Re cloning into home-made T-vector:  Our experience with this is limited to
M13 cloning of PCR products.  It works for us.  As is usually the case with
M13, smaller products clone more readily than big ones.  We have had success
with some inserts >1 kb, however.

I guess the backgrounds (blue and white) are higher than what you would get
with a restricted/dephophorylated vector.  When it works, you see a lot more
whites in the insert ligation than in the no insert control (at least 2x, some
times 10x).  Without seeing the increase over control, there is not much
reason to pursue it.

To improve, I would re-make the vector or re-do the ligation with greater amount
of insert.  Once you get a lot of whites, you do have the option of screening
with one of your primers labelled to identify your clone. I imagine that
increasing the dNTP concentration during PCR might also help to promote extra
addition of A's to the PCR product.  That's a speculation.

Sorry this approach didn't go smoothly for you.  I think it's worth another
try.  To me, the main advantage of this over commercial T-vectors (aside from
cost) is that it allows you (in principle, at least) to use whatever vector
you want.  Good luck.

					Al McGraw
					University of Georgia

					mcgraw%gandal.dnet at server.uga.edu




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