oligo cloning

Jered J Floyd jered at mit.edu
Mon Feb 7 15:40:54 EST 2000


In our lab we generally do 3:1 and 9:1 insert:vector ligations, but I'm
not sure if there's rhyme or reason to those numbers, and everyone I
know does something a little different.  Your numbers seem entirely
reasonable; after CIPing your vector you should have minimized
self-ligation, and you want to avoid multiple copies of your insert
being ligated together.

It's odd that you aren't getting any positive colonies. What sort of
screeing are you performing; colony PCR? Do you get any colonies on a
control ligation with vector alone?  You may want to look at SAP
(Shrimp Alkaline Phosphatase) which can be more reliably heat
inactivated than CIP.


More information about the Methods mailing list