T4 blunting follow by digestion - (Sep/23/2005 )
I want to digest a plasmid with Sal I, blunt the ends with T4 polymerase, then digest it again with SmaI to release a 3.5kb fragment. The intention is to get a fragment with two blunt ends.
My questions is do I need to do an extraction step after the T4 treatment before proceeding with the second digestion, or can I simply heat inactivate the T4 and do digestion straight away?
I suggest to check your buffer compositions to see if they are compatible or not.
I have done many digestions of this sort and have never done an extraction between blunting and digestion with the 2nd enzyme. I heat inactivate my polymerase at 75C for 20 minutes then proceed directly to the next digestion.