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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.Originally posted by dmrodz View PostLeave a comment:
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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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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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