Kpn I site blunting

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Mon Nov 20 22:24:29 EST 1995


In article <30B01433.5073 at mfour.med.kyoto-u.ac.jp>,
hiratamz at MFOUR.MED.KYOTO-U.AC.JP ("Hirata, Masakazu") wrote:

Kpn I cleavage produces a 3' overhanging end not a 3' recessed end;
treatment with Klenow is a poor choice for making it blunt. If you wish to
blunt a Kpn I end, use T4 DNA polymerase in the absence of dNTPs; the
3'-5' exo activity of T4 pol is more active on this kind of template. Add
dNTPs (to 50 uM, final concentration) to the reaction following the T4
cut-back to "polish" the ends.

Alternatively, cut the site with Asp 718 (a Kpn I isoschitzomer) to
generate the 3' recessed ends and then fill-in as per usual with Klenow
and dNTPs.

Cheers

Karl

-- 
Karl Fischer
tyr-2 at bones.biochem.ualberta.ca





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