cloning into plasmid (IMPACT kit from NEB) - (Apr/29/2008 )

I'm having trouble cloning into the vector after digestion with NdeI and SapI. No colonies after digestion. The over hangs are 2 and 3 base pairs long and I ligated multiple inserts for a hour. Will extending incubation time increase ligation efficiency.

-veryslowjoe-

QUOTE (veryslowjoe @ Apr 30 2008, 09:41 AM)

I'm not familiar with that kit, but typically, I'd say yes. If you do extend the ligation reaction time, you'll want to reduce the temperature.

-swanny-

I already run the reaction at 16 C.. do I need to lower it even more?

-veryslowjoe-

ligate overnight at 16 C

-swanny-