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Old 06-06-2020, 02:25 PM   #1
reivan
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Location: Semigallia

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Default SYBR-Gold for 16S library QC

Hi

I'm trying to prepare my first 16s library for ~100 samples and two variable regions, so exciting!!

I've read A Lot of papers, protocols and general chatter from countless talented and experienced people which has brought me to a simple 2-step PCR KAPA hi-fi approach, with 2-10 ng of input DNA, Sequalprep normalization and qubit/KAPA illumina qPCR quantification.

I've played around with most of the involved steps, but the one thing that I've yet to resolve, for my specific setup, is the library size estimation and general QC.
As far as I've read, there are many ways to approach this - people either use Tapestation/Qiaxcel or the good old gel electrophoresis. So this has got me thinking - I am loading a relatively low number of samples on the Miseq (100 samples with 2 variable regions each) therefore I think I could get away with using something like SYBR-Gold to screen for high amounts of nonspecific amplifications, like primer-dimers and approximate the fragment size based on the theoretical size and the corresponding DNA ladder.

Is my rationale correct here, am I missing something? Would SYBR-Gold be an overkill?
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Old 06-10-2020, 07:15 AM   #2
itstrieu
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Default

I don't see why not. I usually spot check with the bioanalyzer after each PCR step to be sure I get the correct amplicon size.
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