1 reply to this topic

[deespike]

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.

[klinmed]

deespike, on Jan 3 2010, 09:58 AM, said:

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 3´termini 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