Klenow Fill-in - How to inactivate it (Dec/05/2006 )

I use Klenow enzyme from NE BioLabs to fill-in. After reaction, The protocol for NE says: add EDTA to a final concentration of 10 mM and heat at 75°C for 20 min.

Could I inactivate just heating? Or do I need to add EDTA always??

I Always do both and then I purify my DNA. I wonder what if I just heat?

I would like to test it, but I don’t have sample and enzyme enough to do it!

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After Klenow, I think that heat inactivation 70°C 20' is sufficient.

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what klenow are you using?

in neb they are the PolDNA Pol I large (klenow) fragment and the klenow fragment (3'-5' exo).

for waht type of enzyme cut?

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DNA Plymerase I, Large (Klenow) Fragment. And I'm using it to Fill-in 5' and Removal of 3' overhangs.

Today, I was looking for information at neb site . and it says:

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.

so, I think I must add EDTA, heat and then, purify. or what do you thinK????

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yes it should be fine

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