We are trying to quantify ammonia-oxidizing bacteria (AOB) and total bacterial (TB) in wastewater. We use gBlcok gene fragments to construct standard curves. For some reason, we rarely get the PCR efficiency in an ideal range (90-110%)? Mostly it is 70-80%. We believe our pipetting is good enough. We make master stock solution with TE buffer and working stock solution & serial dilution with water. Does the low PCR efficiency has something to do with gBlock instability in water or poor primer design? Thank you very much.
Some primers pairs simply do not have high enough efficiency for qPCR. Do you see any sort of pattern for which combos of samples & primers are working?
Other reasons are - degraded template, repeated freeze/thaw of reagents, etc.
Katie, thank you so much for the reply. We have not seen sort of pattern you mentioned. As beginners for molecular technologies, we have used primers that are widely used by researchers. Maybe we could try a few more sets of primers, and see which set works best.
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