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Old 06-29-2011, 01:32 PM   #1
dmrodz
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Location: Puerto Rico

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Default Two emulsion PCRs??

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!
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Old 06-30-2011, 07:25 AM   #2
HMorrison
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Quote:
Originally Posted by dmrodz View Post
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.
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Old 06-30-2011, 08:16 AM   #3
dmrodz
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Thanks a lot
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