phosphatase removal in ligation - (Mar/28/2006 )
we want to do a blunt-end ligation (genomic DNA + linker) and therefore first dephosphorylate DNA with calf intesinal phosphatase (I know SAP seems better, but we have CIP). In the next step protocol says, DNA have to be purified e.g. with a kit to remove CIP. Why? Is it disturbing/killing the ligation reaction in the next step? For us, it would be better to leave out the purification step.
Thanks for help
too fast I found the answer, ligation with ATP and phosphatase doesn't fit...
CIP is a very stable enzyme, hard to destroy and you don't want it to have any activity left in the ligation.
for blunt ends, promega protocols recommends a 2 step : incubate 15' 37° then 15' at 56°. add another 0.01u CIP/pmol of ends and repeat incubation at each temperatures.