CIPping after Klenow blunting - Is CIP necessary in Blunt end ligation? (Apr/01/2005 )

I need to ligate my insert with EcoRI and SalI ends in to Vector with EcoRI ends. I am trying for blunt end ligation in following pattern. Restiction digest both vector and insert, gel elute and purify, NEB klenow (by filling 5' overhag) blunting of vector, CIPping (NEB) the vector, precipitate and setup the Ligation reaction. Please suggest if there is any alternative protocol and also does using of PEG help.... This vector is urgently required .. quick help is greatly cherished.Thank you.

Hi Molonco,

The protocol looks alright but make sure you PNK treat your insert after Klenow treatment.

Also an alternative could be to do a two step ligation protocol whereby you CIP treat your vector and then ligate your vector/insert using the solitary EcoR1 site on the insert. After first ligation you Klenow treat the product and ligate again, this is particularly useful if you are using a large insert that doesn't want to go in with blunt cloning. Also to reduce background after the first ligation you can gel purify the 1st step ligated product away from the vector alone. I have success doing this when everything else has failed.

Cheers,

Scott

hi
scott is right you definitely need to phosphorylate your insert when CIPing your vector.
But i'm wondering why don't you perform a classical ligation as your restriction sites are not compatible?