pGEM-T Easy - how long can the insert be? - (Feb/27/2008 )

Hi all,

I have a 6.7 kb PCR product which I tried to clone into pGEMT-Easy vector. It didn´t work (i tried several times). Can it be due to the size of the PCR product? I use pGEMT for circa 3.5 kb PCR products and it works good. I´m pretty sure that the vector:insert ratio was correct (calculated according to the instructions). The competent cells are O.K.
Should i try again or should I use a different vector?

Thanks for replies.

I think you should try again. I've cloned 6kb inserts in pGEM-T Easy very easily. Are you sure that your polymerase adds dATPs at the 3' ends of your PCR?

what do you mean my didn't work...
were there no colonies on the plate?
was there too much background on the control plates?
did the colonies you picked not have the instert?

there are many way's things do not work.

V

ok the redo 10' at 72 with 0.5µl fresh Taq polymerase and redo the procedure.
what ratio (plasmid / insert) did you try ?