Gel-size selection in NimbleGen exon capture

[mitchelS]

Hey,

just wondering if anyone has tried omiting the gel-size selection following Illumina adaptor ligation prior to commemcing the LM-PCR step in the NimbleGen in-solution Exon capture protocol?

If so does the residual adaptor dimers have any marked effect on the downstream data?

Cheers,

Mitch.

[Liting]

Hi，mitchels
I am tring the gel-size selection following Illumina adaptor ligation prior to commemcing the LM-PCR step in the NimbleGen in-solution Exon capture protocol。But we can't get enough PCR products after LM-PCR（8 cycles）。Are you successfully with the protocal？
Thank you！

[apeterson]

I have found that the skipping the gel size selection can result in increased primer dimers in the pre-hybridization PCR. The Illumina protocols suggest an extra magnetic bead clean after ligation, this seems to help with the PCR.

[csquared]

Size selection is definitely not needed by gel, but a bead-based size selection to get rid of adaptor dimer is really helpful. A single 1x bead purification will get rid of stuff below 200nt prior to library amplification.