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Hi y'all, I left my complete sequencing reaction at room temp right outside the thermocycler by accident for about ~45 min b/c I was waiting for the lid to heat and forgot about it. It's the normal Big Dye terminator sequencing rxn, forward or reverse primer, big dye, water, sequencing buffer. I found my mistake and quickly put it into the PCR machine. DO you think it will be ok? I do. Of course BDTv3.1 is light sensitive but it was kinda in the shade, not under direct lights.. I hope 45 min isn't too long. Thanks!
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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...