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Background: made overlap PCR construct of 3.4 kb, final destination is a home-made agrobacterium transformation vector. I am trying to clone the product into an intermediatory vector in order to increase the amount of DNA I have to play with (and hopefully get cleaner DNA than from a PCR and consequent gel extraction).
So far I have tried cloning the amplicon into pGemT-easy and pPCR Script, both backbone vectors are ~3kb. I have abandoned pGemT for the moment and am trying to work in pPCR Script. What I am finding is that only part of the amplicon is getting put into the vector - you can see this due to the unexpected length of the construct when run on a gel - also I have sequenced several of the damn things and I am finding only bits and pieces have been stuck in - in one colony the whole centre bit of the construct had dropped out but the two ends were perfect!!
I am transforming the ligations into E.coli that have had their recombinatory mechanisms removed - so that can't be the issue.
Rather than spend even MORE time trying to figure out what could be happening I am wondering if anyone has a suggestion for a cloning vector that reliably incoporates PCR amplicons >3kb? I know several of the product guides SAY their vectors take up to 10kb - but if anyone with experience could recommend one that would be great. I have been looking at CloneJET by Fermentas - any recommendations would be MUCH appreciated!!
Cheers, A.
