blunt cloning of PCR fragments

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Mon Jul 31 21:36:11 EST 2000


Yeah, why not give polishing w/ Klenow a try. It's only a 15min reaction...

At 05:04 PM 7/31/00 +0100, Michael L. Sullivan wrote:
>I've been trying to blunt-end clone some PCR fragments without much
>success.  Controls lead me to beleive that there isn't  problem with the
>ligase or ligation condition.  The PCR fragments were generated using
>KlenTaq LA from Sigma.  Since the enzyme mix is supposed to have a 3' to 5'
>proofreading activity, I figured the fragments should be coming out of the
>reaction with blunt ends.  Am I wrong about that?  Should I go ahead and
>polish up the ends in a separate step anyway?  Thanks for your input.
>
>Mike
>
>Michael L. Sullivan, Ph.D
>
>U.S. Dairy Forage Research Center
>1925 Linden Drive West
>Madison WI, 53706
>
>(608) 264-5144 Phone
>(608) 264)-5147 Fax
>
>
>---
>
>
>
Dr. Hiranya Sankar Roychowdhury
Dept. of Molecular Biology	
PO Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


---






More information about the Methods mailing list