Sticky-end dephosphorylation - (May/18/2007 )
Please, help me.
I have two questions. Does phosphorylation step needed to be done only in the ligation with the blunt end restriction enzymes? Would it help if I phosphorylate the sticky-end RE?
Thank you.
I have two questions. Does phosphorylation step needed to be done only in the ligation with the blunt end restriction enzymes? Would it help if I phosphorylate the sticky-end RE?
Thank you.
We donot dephosphorylate when we do sticky end digestion provided the ends are not compatible.
For all blunt ends we dephosphorylate the vector
Both blunt end ligation and Sticky-end ligation with compatible ends need vector dephosphorylation.
However as scolix has mentioned, sticky end ligation with non-compatible ends do not need dephosphorylation.
Nevertheless in good hands, vector dephosphorylation does help decrease the background, especially when working with very large vectors >10kb. Unfortunately it is rather easy to over dephosphorylated and damaged the ends of the vector, making said vector unligatable. So dephosphorylation can be more hinderence then help.
if you dont dephosphorylate you vector with compatible end you will got all self ligated plasmid