Ligation and transformation problem - (Jul/30/2007 )

Hello everyone
I am trying transformation of 1.5 kb pcr product in 3 kkb PGEM-t Easy vector..........since two weeks...but not getting positive clones...
everytime..I am getting some white colonies but none of them found positive.
I am using new ligase , for ligation and Insert-vector ratio is around 1:3 ....
where should be the problem?
suggest me............i am getting frustation ...............

Thanks in advance............

If your pcr polymerase is proof-reading you may not get the A-overhangs. I don't know PGEM-t Easy vector, is it a TA-cloning?

If that's not the case, are you trying blut-end cloning ?
There's the possibility that your fragment (and plasmid) are self-ligating.
I knew some people who had problems with cloning fragments over 1kb because of that, and only after many transformations did they get one colony.

If that's your case I'd say try to get some cohesive ends...

Did you check the PCR on gel? Are you digesting the PCR product or is it TA cloning?

How are u calculating insert vector ratio? did you take size into account? Regardless I would try for 3:1 instead... I have successfully cloned inserts from 1.5-2.5kb into T-easy, I get a good strong PCR product and add as much as possible to the ligation reaction...

of course suggestions above too, you have to get the A overhang with your taq for T-easy cloning to work...

HTH and good luck

i think the insert to vector ratios work best at 1:1 to 3:1, but pGEM-T vector can work from 1:8 to 8:1

by thw way, how do you screen your white colonies?