oligo cloning

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


Caroline,

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.

--Jered




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