Cloning a 17 kb contruct - (Apr/19/2015 )

Hi!

I'm trying to subclone a contruct from pBluescript to a agrobacterium vector (this one is ~11 kb) using the sites PuvII from the pBluescript and SmaI from the agrobacterium vector (both blunt ends). the final construct will be 17 kb. I tried a lot; but I never get colonies on plate.

I'm using e.coli top10 cells and using heat-shock as transformation method.

what is going wrong? any suggestion? 

thanks in advance :)

Most likely: competent cells are low efficiency

Next: Bad DNA (impurities, not there, UV damaged)...

Next: Ligation conditions (buffer bad, too much DNA)

For a 17 kb plasmid you'd better use elecro-competent cells, not chemical competent cells, specially if you made the chemical competent cells yourself in the lab. Home made competent cells are not always as efficient as you expect them to be.