Ligation

Deitiker, Philip R via methods%40net.bio.net (by pdeitik from bcm.tmc.edu)
Thu Jun 21 16:09:50 EST 2007


 

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Tim Fitzwater
Sent: Thursday, June 21, 2007 10:14 AM
To: methods from magpie.bio.indiana.edu
Subject: Ligation 

"
They are usually used at high temperatures because 
that inhibits (but does not eliminate) the contaminating
exonclease activity.
"

How high is high temperature? 45'C, 55'C, 65'C

"
When using these enzymes, I dilute
the CIP/BAP as recommended for the number of ends (sticky
or blunt) and react at the temp and time recommended.  
"

37'@ (1U CIP/2ug DNA)/hr? 

"
I then clean the DNA thoroughly and repeat the CIP/BAP one
more time
"

37'@ (1U CIP/2ug DNA)/hr? 

"
 because the reaction is self-inhibiting and does 
not go to 100% the first time.  SAP is much more forgiving 
and can be used simultaneously with the restriction enzymes.  
"

I have been using CIP with REs as this is what was recommended
1U CIP per 5 ug/DNA for 3 hours at the end of the reaction. The last
couple of times I did longer because of the excess of vector closure. 
NEB says you can use CIP in NEB 2,3,4,EcoR1 and BamH1 buffers. 

How does one know whether the CIP has worked and the ends are still
intact? T4 PolyNt kinase? 

I have to think about this abit. Thanks. 



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