TA cloning-Insert size too big? - (Feb/26/2006 )

Hi, I am current trying to clone a PCR fragment of 2.1kb into pGEM-T easy (3kb). Have tried a few times without success (little or no white colonies). The ligation buffer and ligase are both fine (I did a different ligation and it worked fine). The vector is brand new. and there is no apparent fault with the insert (purified and amount judging from the gel is OK). I am wondering if the insert is too big for the vector to handle. I should mention I have cloned two 1kb genes using the same method successfully (so no exonuclease chopping off the A). Can anyone shed some light on this? Thanks

The insert is probably not too big for the vector. I would try different insert:vector ratios. For me, 5:1 - 3:1 worked well. Make sure your bacteria are competent and the insert has an A-overhang.

i've cloned 7kb in such vector