Adding A overhangs - (Jan/29/2008 )

Hello,

I usually perform this A overhang reaction before performing a ligation with the TOPO TA cloning kit:

10 ul PCR product
5 ul 10x PCR buffer
1.5 ul MgCl2
1 ul dATP
1 ul Platinum Taq

I then proceed to the ligation, and electroporate.

I need to repeat the ligation but I do not want to go back to my original PCR product - how can I repeat this A overhang reaction?
Can I just treat the A overhang reaction as my PCR product by adding 10 ul of it and then adding the buffer, MgCl2, dATPs and taq?

Seems logical, but I'd love some more expert opinion - thanks!

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Why don't you just use a polymerase, that adds A overhangs naturaly during PCR (all Taq derivates does) of mix if you need to use proofreding enzyme too?
You only add like 10 minute elongation step on the end of the reaction to be sure all products have overhangs.
And then use a fresh product for ligation, it's always better than use stored product.

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Thanks for the advice!

Well, Im trying to stick to the polymerase I'm using now. I can't order anything else.

Also, the PCR product was not created by me. It is a sample received from collaborators - hence I want to preserve it and I dont have fresh product. The A overhang reaction Im doing is essentially a 10 min elongation at 72C.

I'm most curious as to whether I can substitute my initial A overhang reaction for 'PCR product' in this recipe:

10 ul 'PCR product'
5 ul 10x PCR buffer
1.5 ul MgCl2
1 ul dATP
1 ul Platinum Taq

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yes, it should be possible as long as your input PCR DNA has been desalted.

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