Am I in need to A tail my amplicons before adapter ligation

[ghanim]

Hi
I am planning to amplify short amplicons (~150bp) and then ligating the adapters (so as to skip the enzymatic fragmentation), I am just wondering whether should I do A tailing to the PCR products prior to proceed to adapter ligation or not??

Thanks

[nucacidhunter]

It depends on the polymerase used for amplification and type of adapters. If you have used proof reading polymerase and adapters have T overhang it will be necessary.

[ghanim]

Thanks,
I am planning to work with user-defined protocol... so I am not aiming to include the T overhang to these adapters and use the Hircullase polymerase ( high fidelity)...So, in this case, I don't think I need to add A tail
But the problem is that this overhang is important for efficient adapter ligation...isn't it?

[nucacidhunter]

T overhang in your adapter will increase ligation efficiency if you add A tail to your amplicons. By using that polymerase you will need to clean up your PCR reaction properly if you want to proceed to A tailing to avoid 3' to 5' exonuclease activity of residual proof reading polymerase. I do not know which user-defined protocol you are using.

[ghanim]

I agree, SPRI need to be done... I am planning to work with a modified Nextera protocol..
Thanks