Dephosphorylation for one cohesive, one blunt end ligation? - (Jul/18/2007 )
HI, I am a novice in molecular biology and just want some consultation before proceeding
I want to subclone an insert into a vector.
Insert is cut out as follows: 1 end with EcoRI, one blunted (with Klenow after XbaI)
Vector is cut as follows: 1 end with EcoRI, one blunted (with Klenow after SalI)
If I put both together into a ligation mixture, what are the chances that they will ligate correctly (EcoRI with EcoRI, blunt with blunt)? Can the vector religate in this case? Do I need to dephosphorylate either the vector or the insert before performing ligation? How much of T4 ligase should I use? Thanks for your help!
you should do ecoR1 digestion in second.
the chances that the vector religates comes more from the remaining single cutted vector that regulary remain in extra little amount (but still poison the ligations )
so dephosphorylate the vector with the procedure for blunt ended extremities, becuse the procedure is generally longer, but more powerful too.