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can klenow fragment be used to remove the 3' overhangs?


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#1 deespike

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Posted 03 January 2010 - 12:58 AM

Hi all

I have a query regarding the use of klenow fragment - Can the klenow fragment from different companies be used to remove the 3' protruding ends ? beacuse in neb catalog its mentioed that the klenow fragment can be used but other companies donot mention it.

#2 klinmed

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Posted 03 January 2010 - 08:37 AM

Hi all

I have a query regarding the use of klenow fragment - Can the klenow fragment from different companies be used to remove the 3' protruding ends ? beacuse in neb catalog its mentioed that the klenow fragment can be used but other companies donot mention it.

Klenow (from any good manufacturer) can be used to remove protruding 3ītermini but traditionally T4 polymerase has been preferred. However, klenow has extendase activity and this can significantly lower subsequent cloning efficiency.
I usually use a thermophilic proof-reading polymerase (pfu, pfx etc) for all blunting. Just incubate your fragment for 10 mins at 72 oC in 100 ul x1 buffer + 10 mM dNTPs + 1 U enzyme.

You may find this paper of interest:

Costa, G.L. and Weiner, M.P. (1994)
"Polishing with T4 or Pfu polymerase increases the efficiency of cloning of
PCR fragments" Nucleic Acids Res. 22, 2423

Hope this helps




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