blunt end ligation after inverse PCR - (Nov/23/2006 )
I am using IPCR to incorporate mutations into a gene which is cloned into pUC18. I have phosphorylated the primers. After PCR i get the expected linear product. I then add T4 ligase and buffer and ligate overnight at room temp. However after transformation into E.coli JM109 i do not get any colonies. Both my +ve and -ve controls work. Does anybody have any ideas?
What polymerase are you using? Are you digesting plasmid with dpnI?
The + and - controls, are they the whole procedure (PCR, ligation, transformation) or just transformation?
I am using Pfu in the PCR. I have done the experiament in duplicate, with one of the PCR amplified products i have used Dpn, the other i have not.
The controls used are just for the transformation procedure.
Blunt end ligations are often problematic. What enzyme are you using for PCR? You may be producing A overhangs which will inhibit blunt end ligation. A blunt ligation should normally be done at a lower temperature than RT. It will help a lot to have PEG in your ligation buffer, found in kit form in the "Quick Ligation" kits.
If I were doing it, I would rethink the strategy and go for offset cutters in the 5' tail of your primers, or choose another way to insert your mutation.
about 10% PEG 6000... magic ingrediant in the quick ligation buffer
The primers are phosphorylated yes?