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