i have a question concerning the efficiency of ligations.
Is it significantly higher efficient to ligate NOT dephosphorylated sticky ends compared to SAP treated ones (Vector only, Insert not)?
If somebody of you knows, how much/which order?
I'm asking because we have a problem with subcloning, VectorA (7kb) with InsertA(1kb), VectorB (4kb) with InsertB(1kb); i.e. we try to clone Insert B in Vector A, Insert A and Vector B are "waste". However, we have really low yields of DNA and in Gel extraction steps we lose nearly all Insert. When we now Dephosphorylate Vector A (incl. Insert A)and add Vector B (incl. Insert in relative excess, i.e. as much as we can, is it possible to get VA-IB clones? In an acceptable ammount? Or should we dephosphorylate the other way round?
If the efficiency of ligation of normal vs. SAP treated is the same, we should get a 1:1 ratio of clones (VA-IB : VB-IA), don't we? But if the efficiency is not the same, we get more backligations, i.e. VA-IA.
Thanks for your help
Efficiency of SAP treated Plasmid Ligation
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