4 replies to this topic

[claritylight]

I have DNA that already went through blunt end. I want to do extension with Klenow exo- and dATP, then ligate some linkers. The Klenow exo-/dATP step will add A base to the end of my blunt ends. Then I can easily ligate my linkers which have a T base on their ends.

My question is, what happens theoretically if I add in excess of Klenow exo-?

[NemomeN]

Don't really know that answer about excess Klenow, but you can get the same affect by adding in Taq polymerase...and just follow the normal PCR conditions (dATP, buffer, taq, etc) and do an extension at 72 for 20 min.

[claritylight]

Correct me if I'm wrong, but Taq polymerase will add 3'A base by itself or does it have to do it in the presence of dATP?

What would happen if you add in excess Taq especially to the ligation step?

[WHR]

claritylight, on Mar 1 2009, 07:42 PM, said:

Correct me if I'm wrong, but Taq polymerase will add 3'A base by itself or does it have to do it in the presence of dATP?

What would happen if you add in excess Taq especially to the ligation step?

dATP is required.
Excess Taq will not affect ligation.

[claritylight]

WHR, on Mar 2 2009, 08:09 AM, said:

claritylight, on Mar 1 2009, 07:42 PM, said:

Correct me if I'm wrong, but Taq polymerase will add 3'A base by itself or does it have to do it in the presence of dATP?

What would happen if you add in excess Taq especially to the ligation step?

dATP is required.
Excess Taq will not affect ligation.

Will it affect anything in the extension step if excess Taq is added? Meaning, when the dATP runs out, Taq will just stop?