T4 DNA polymerase blunting

T. S. Pillay tpillay at ucsd.edu
Thu Feb 24 01:15:28 EST 1994


Subject: T4 DNA Polymerase for blunt ending cDNA
From: Melissa Phillips, bjweis at iastate.edu
Date: 12 Feb 94 20:26:32 GMT
In article <bjweis.761084792 at pv0a0c.vincent.iastate.edu> Melissa
Phillips, bjweis at iastate.edu writes:
>Hello:
>
>I am having problems blunt ending my cDNA with T4 DNA polymerase.  The
>enzyme is not very efficient in my hands.  The T4 I am using is from a
cDNA
>kit from Bohinger Manneheim.  I am considering running the reaction once
with
>the dNTPS in the reaction mix, and then running it again without them in
order
>to utilize the exonuclease activity.  Has anyone out there had any luck
doing
>this, or can you recommend any other  way to increase the efficiency of
the reaction.
>
>
>Thanks,
>Melissa Phillips
>Iowa State University
>-- 


Subject: T4 DNA Polymerase for blunt ending cDNA
Mellissa
Have you succeeded in blunting your DNA yet?
What conditions are you using ?
I've found that using T4 DNA poly at 12 degrees C and optimal dNTP
concentrations , and using buffer H from Boehringer works best for me. I
don't have my book here but can send details if you so require (See Short
protocols in Mol Biol for dNTP conc).  I've used this to "trim" PCR
products prior to blunt end cloning. Klenow does not work well in my
hands for 3' A overhangs

T.S. Pillay
tpillay at ucsd.edu  
Tel:  619-552-8585 ext 7148  Fax:  619-534-7181



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