PCR conditions for PE adapters

[Luke]

Hi,

I was wondering if anyone could help with the optimal cycling conditions for the post-adapter-ligated amplification step (using PE adapters) in the Sure Select Target Enrichment system? Given the short and long PCR primers required, is a sort of 2-step PCR required?

Additionally if one were to use the "homemade" adapters, are the adapters required to be pre-annealed before the amplification? How are these "Y" -shaped adapters generated?

Any help would be great,

Thanks,
Luke.

[DPB]

Hi Luke, I'm also looking for PCR conditions for the post-adapter ligation step. I ordered single stranded HPLC purified adapters and annealed them before ligating to the sequence. I'm not sure how it has worked yet as I haven't done the PCR.

[Luke]

I recently tried annealing the adapters together, amplifying, and running the products on a agarose gel.. i obtained 2 amplification products... one approximately 150bp and one slightly smaller.. i've gel extracted these bands but whether they do represent genomic-adapter fragments remains to be seen.. I'll keep at it, and post any progress.

Cheers,
Luke

[terri]

y-shaped adapters

Hi Luke,

have you made any progress with creating the y-shaped adapters?
We sequenced them ourselves as well and I wonder if a preannaeling
process is necessary because Illumina supplies them in a mix already.
So I have the same issue than you have.
Did you recieve any information yet?
Thanks!!!!!
Theresa