Two emulsion PCRs??

[dmrodz]

Hi!!

New to the 454.

I need to know this: for shotgun sequencing, if I want to do a bulk run: do you do 2 emulsion PCRs??? One with the SV kit, to do the titration (up until % enrichment bead calculation) and another 6hr amplification for the bulk run (with the library quantities obtained from the titration enrichment?

THANKS A LOT!

[HMorrison]

Hi!!

New to the 454.

I need to know this: for shotgun sequencing, if I want to do a bulk run: do you do 2 emulsion PCRs??? One with the SV kit, to do the titration (up until % enrichment bead calculation) and another 6hr amplification for the bulk run (with the library quantities obtained from the titration enrichment?

THANKS A LOT!

That is certainly the safest way to go. In our experience, starting at 0.5 to 1 cpb works most but not all the time and it's sad to waste a large volume emPCR kit and get 35% enrichment. We do 8 SV reactions at 4 input amounts.

[dmrodz]

Thanks a lot