I try to detect bacterial 16S rRNA gene by using probe based qPCR. I found that my negative control (no template) has positive signal at late cycles (Ct>35). this result was confirmed by using new probe, new primers, new ddH2O, new master mix reagent (KAPA probe fast qPCR kit) and new assay (droplet digital PCR). my colleagues suggest that It caused by DNA contamination in qPCR reagent during Taq polymerase production from company. How do I resolve this problem ?
Note, shrimp nuclease can use to remove dsDNA in general, it possible or not if i use it to treat taq master mix reagent prior to mixing with the sample ?
Wouldn't carryover nuclease in the PCR mix also degrade your cDNA template after reverse transcription?
It could also be environmental contamination (from the air, surfaces, etc.). You should assemble PCR master mixes (primers + buffers + enzyme) away from your template - ideally in a laminar flow hood in a different area in the lab than where bacterial work or DNA preps are done. I would also recommend filtered pipette tips if you're not already using them.
>35 cycles is very late. I wonder if it's even worth worrying about - if your samples are hitting threshold 10 cycles earlier, then the contaminant is comprising only ~0.1% of the input cDNA.
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