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Is PNK treatment required for my insert? - Is PNK treatment required for my insert after i CI (Apr/23/2005 )

In my cloning expt after blunting vector and insert I CIP my vector alone. Then to carry out blunt end ligation do I need to phosphorylate my INSERT? How could I increase the efficiency of ligation apart from using PEG 4000 and keeping O/N? Any Suggestions would be of great help!


normally blunting ends do not dephosphorylate DNA. The basical thing that may improve efficiency is to increase amount of insert.
I've tested several protocols. One to 4 hour at 25° or overnight at 14 or 16, tried quick ligation kit...
But basically you should dephosphorylate your vector and phosphorylate your insert.
i can't tell you much more...


I think phosphorilation of the insert is essential, dephosphorilation of the blunt end vector is not essentail but preferable, specially if the insert is of large size, why dont u tr both and see