3' adenine overhangs - PCR products

[burkx]

Hi,

I'm currently planing to clone a bisulfite converted DNA fragment. It's the first time we do this type of experiment in this lab. I bought the TOPO(r)TA cloning kit from Invitrogen to insert a 100 bp fragment in a TOP10 vector (I know I'm not using the template, but since I haven't tried it yet, It is hard to fill it all out).

I'm using relugar Taq polymerase.

I was wondering, how do I know if my products have 3' A overhangs? or if they're blunt ended?

Do I have to design the primers especially for that? or do I simply assume they are blunt and proceed to a protocol to add the A overhangs?

Thank you for your time
Regards, Burkx

[relaxin]

Regular Taq will add the 3' adenine overhangs for you. I do not know how efficient this process is, as I never use the T/A cloning system.

[burkx]

Thank you very much,

Regards, Burkx

[mchlbrmn]

I've done it a long time ago.
My recollection is that I may have used an extended final 72C incubation to encourage the adding on (but I don't know if it helps). Other than that, the taq should do it.

[r.rosati]

Taq adds an A (or another nucleotide, or nothing) depending on the last nucleotide of your product.
I always like to refer to Tim Fitzwater's post here. In my opinion It's the best post in this Forum about A overhangs.