as i used a super high fidelity DNA polymerase to finish PCR and then extract my desired length from the gel ,now i want to use an other 2 × pcr taq Master mix to add A Tail to my desired length of DNA can is it possible to add A Tail by doing this.
procedure :
5 μl pcr recycled product.
5 μl 2 × pcr taq Master mix
then put it in centrifuge machine at 72 ºC for 15 minutes
my question is is it work or not.
In essence, yes I think that should work
Poly A over hangs are usually provided by incubating DNA with Taq @ 72C for anything between 10 min to 30min
It should work but if it didn't, then i think you need to gel purify your PCR product to get rid of any proofreading enzyme present which can interfere with the addition of polyA overhangs.
Alternatively if you need just a single A overhang, you can use ExTaq DNA polymerase from TaKaRa in first place itself. It adds a single A overhang and also have a proofreading activity.
Hi Fahim
The protocol I use is a bit different. I take my PCR product and I clean it using a spin column. Than I combine, PCR product with Taq, buffer and dATP for 72oC for 10min.
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