Klenow - (Mar/05/2007 )

Hi there,

can you tell me whether I can use Klenow to create blunt ends in my vector, then heat inactivate it and add the next RE without purifying the Klenow-treated vector in between? On NEB website they say Klenow works in all NEB RE buffers...

Thanks a lot, Isabelle

NEB FAQs for Klenow Fragment:

Q6: Can DNA Polymerase I, Large (Klenow) Fragment be heat inactivated?

A6: Yes. Add 10 mM EDTA to chelate the Mg2+ cofactor, which protects the DNA ends as they "breathe" while the temperature is increased. Then heat at 75°C for 20 minutes. (Heat inactivation in the absence of EDTA accelerates the 3' > 5' exonuclease activity.)


Q7: Are the nucleotides needed to remove a 3' overhang with DNA Polymerase I, Large (Klenow) Fragment?

A7: Yes, in the absence of dNTPs the enzyme will remove more nucleotides than necessary to blunt.


Q8: What are the main causes for blunting reaction failure using DNA Polymerase I, Large (Klenow) Fragment?

A8: The following conditions can cause the exonuclease to overwhelm the polymerase activity causing recessed instead of blunt ends:
*Adding too much enzyme
*Incubating for too long
*Incubating at temperatures greater than 25°C
*Failure to add nucleotides or the nucleotide level is too low
*Heat inactivating without EDTA

If you use another Klenow from NEB (Klenow -exo), you don't have the exonuclease activity, and after adding the dNTPs and filling up your 5' overhang, you can heatinactivate it (never had problems with heatinactivation w/o adding EDTA) and then digest your DNA with the next RE.

aztecan princess: I use NEB Klenow -exo
Here's the link: http://www.neb.com/nebecomm/products/productM0212.asp

Never observed 3'-> 5' exonuclease activity...