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Old 08-16-2017, 11:59 PM   #5
rudzik79
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Location: Poland

Join Date: May 2016
Posts: 10
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Wow, Adam, thank you very much for such a nice response and willing to get to the bottom of this. I am afraid I got a simple answer yesterday, and it isn't pretty.

It was the primers all along.

I asked my colleague from another institution to come with her primers. We compared the sequences, they were identical. Nobody made any errors in sequences during purchase.
So we ran four parallel PCRs - iProof and KAPA, with her primer pair and with mine.
The results were striking, as hers worked perfectly in both mixes, on the same PCR program. Samples with my primers were, as usual, empty.
I am doing this for 17 years, and was in institutions that have ordered thousands of primers by now. NOTHING LIKE THIS has ever happened, except for my very first pair - but back then the sequences were copied by hand, and a lady from purchase simply made an error in polyN stretch.

I will be filing a complaint and demanding compensation for lost time and kits. By the way, my NEBNext kit is finished and I am really furious.
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