problem about vector Dephosphorylation (View forum version)



tantao

Posted 13 July 2010 - 08:19 PM

Dear everyone,
I have questions about the vector dephosphorylation and ligation. When I digest the vector with enzyme, in order to avoid the vector self-ligation, the vector was dephosphorylate. Then I digest the insert fragment from another vector and ligate them. But I think maybe there is problem, the insert and the vector only form two phosphodiester bonds and the other two sites are nicks, they can not form phosphodiester bond. if when this product transfects into the E.coli, the bacteria will repair these two nick? Could you give me some suggestions, Thanks in advance.

molstudent

Posted 13 July 2010 - 10:17 PM

on the NEB website for T4 DNA ligase it says this in part of the description:

"...This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1)."

tantao

Posted 14 July 2010 - 01:13 AM

on the NEB website for T4 DNA ligase it says this in part of the description:

"...This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1)."



Thank you very much molstudent!