ACK! Blunt end cloning - I'm confused.. - (Sep/11/2005 )
Ok I am trying to make a library.. I am digesting my bacterial genomic DNA with: Sau3A1 (5'...*GATC ...3') and am digesting my vector with BglII (5'...A*GATC T ...3')
shouldn't i need to treat my vector with something to remove the overhangs in order to do a blunt end ligation?
someone told me no, even though i suspect yes, and my ligations aren't working.. suggestions??
i should treat with klenow right? to fill in the overhang and make a blunt end? anyone?
I have had good luck with Klenow in the past (and yes, to get any sort of efficiency at all, you need compatible ends!)
NEB (new englad biolabs) is a fantastic resource...Promega also has good stuff on their website...you probably want Klenow because it's pretty straightforward and relatively cheap, but there are a couple of other options.
If you have one end blunt, ofcourse the other end should be made blunt:
5'-overhang should be filled in with nucleotides
3'-overhang should be removed
both reactions can be performed by klenow, but the difference is adding nucleotides in case of filling and leaving the nucleotides out of the reaction mix in case of removal.