cloning, ligation, dephosphorylation - ligation, dephosphorylation (Oct/23/2007 )

Hi,

If vector and Insert both are double digested, are they have to be both dephosphorylated?

If insert was prepared from PCR where this insert was used as template too, is this PSR pruduct need to be dephosphorylated?

as a rule only the vector need be dephosphorylated.
If you dephophorylate both vector and insert, neither DNA strand will be able to ligate.

if the ends of your vector are non compatible @ vector has been cut by BamHI and HindIII, dephosphorylation is not an exacting requirement.

In fact, done incorrectly (too much CIP, too little DNA or too long) dephosphorylation will damage the ends of your vector rending it unligatable. Thus dephosphorylation can at time be more trouble then it is worth and some people do not dephophorylate unless they have too (such in situations where the vector has compatible ends, vector cut once by EcoRI)

We do not dephosphorylate PCR products or any insert. Its only for the vectors if there are blunt ends.