methylation affects ligation? - (Jul/03/2008 )
I have this problem.
I cloned a 1040pb-PCR-fragment into pJET. Now I need to cut out my fragment with ApaI and NgoMIV.
It was a little difficult to cut with ApaI because my construction has an overlapped methylation site with ApaI so restriccion is partialy blocked. To solve this situation I used more enzyme and more time. Finally I got my fragment of 1016 flanked with ApaI and NgoMIV with no further problems....
But now my NgoMIV/ApaI fragment wont ligate to my vector (a 2634pb fragment that comes from pBSKII+) ...
I've already tried 1 hour ligaton RT, 16 hour ligation 4°C, 16 hour ligation 16° with out results.
Do you think the methylation overlapping the ApaI site can affect ligation???
Hallo, I am not sure I have full understood your suggestion
1. you cloned this PCR fragment into pJET cultivate in bacteria, purified plasmid and cut. What kind of the host strain did you use. commonly used host strains as XL1, DH5 alpha, Novablue suppose to be restriction methylation deficient since genes for this system are disrupted. But is is not it is pretty possible methylation will bloc ligation.
You can buy demethylase and treat your fragment before ligation. But first check your host strain
good luck
1. you cloned this PCR fragment into pJET cultivate in bacteria, purified plasmid and cut. What kind of the host strain did you use. commonly used host strains as XL1, DH5 alpha, Novablue suppose to be restriction methylation deficient since genes for this system are disrupted. But is is not it is pretty possible methylation will bloc ligation.
You can buy demethylase and treat your fragment before ligation. But first check your host strain
good luck
Yup... I cloned the PCR fragment into pJET, cultivate, purified and cut. I used DH5 alpha as host, but I still think that my fragment could be methylated because of ApaI deficient cut.