Generating "good" blunt ends

Mike Dalrymple dalrymple at pplros.demon.co.uk
Tue Nov 21 06:46:10 EST 1995


In article <48pi33$2cl at infoserv.rug.ac.be> Nino, nivil at gengenp.rug.ac.be
writes:
>I cloned some cosmids randomly in M13 to generate DNA sequencing, we shear
>these cosmids by sonication and lately by nebulizing the DNA, afterwards we
>made a DNA ends repair by using T4 DNA Pol. and Klenow enzyme and we separate
>according to MW. by agarose gel. (~1100 bp.)
>Our control is with lambda DNA digested with AluI enz.
>My problem is the low efficiently of cloning after T4 and Klenow treatment.
>Any suggestions are WELLCOME.
>

Why are you using T4 AND Klemow?  I use T4 for blunting 5' and 3'
overhangs
in cloning, and it works fine. I use it in the restriction buffer or in
Boehringer
'A' buffer, and add 200 micromolar dNTPs.



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