Self-made TA vector

ma ddsyr at PUBLIC.EAST.CN.NET
Wed Sep 16 03:31:14 EST 1998


Dear Netters:
I saw the discussion about self-made TA-vector. so Iike to talk my
experineces. I make the vector by cut with SmaI of pGEM vector then add
the T by Tag. The add seem OK since when I self ligate this vector, it
cannot get the clone by transform the DH5, before add T of course it
can. But when I add the PCR products  and ligate to this T vector, I
cannot get any clone by transform the DH5, even I put the final
extension time to 30 min in PCR( one report in Biotechnique say this
will help to increase the overhang A in PCR product). So I ligate my PCR
product into the blunt pGEM (just SmaI cut), I got some clone, although
some clone are not right insert. I guess maybe the T-vector has more
than one T or PCR product has more than one A? But I have no evidence.
Anyone have this experience please post your valuable ideas.
Dr Q. H. Ma
Institute of Botany, Academia Sinica
Beijing 100093, China





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