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10x Library Prep..unusally low amount

Posted By: Hedgehog_, on Aug 20, 2018 at 3:29 PM

Hi there,

 

I just generated 4 10x chromium scRNAseq libraries (V2 3' scRNAseq). After cDNA stage, I got a range of 80-230ng of cDNA. they were all looking good. But when I generated the libraries (used 12 PCR cycles), I got about 1-2ng/uL back...I feel like this is unusually low as I am expecting 40-44ng/uL (Qubit quantitated). I thought I had done most of the steps...has anyone come across this before?

 

I will do a qPCR on these libraries using the Kappa kit to see if they can be sequenced. Anyone seen this before...I checked all PCR programs and ticked off every reagents that need to be added but guess I have missed something......important...

 

Let me know if this has happened to you before....

Thanks heaps!

 

Hedgehog