Even after multiple attempts using fresh master mix, probes and primers, the international standard continues to fluctuate in efficiency from 80-96%. Obviously I need it over 90%. Does anyone have any other suggestions on what may be causing these inconsistencies? The reagents have been prepared to aliquots to minimize freeze/thawing and each run is in triplicate from 10^7-10^3 dilutions.
Hello Jessica
I had similar issues..which I sorted out lately
Dnt mind me asking, could you elaborate a bit, what are you trying to perform here?
Plus is it based on SYBR chemistry?
You're making a new standard curve from each new aliquot - are you using the same pipettes for each dilution series? Is it volumes states in the SOP - or the dilution factor required? (ie are some using 2 +18ul and others using a (usually) more accurate 5 +45ul, if the SOP says 'make 1:10 dilutions').
How is the standard stored? In aliquots? Fridge or freezer? silconised tubes?
I am trying to measure parasite levels in blood using FAM probes. It is the one international standard from WHO which I extracted the DNA and re-suspended it in elution buffer. It's stored in low-bind eppendorf tubes at -20deg not in aliquots.
I am using the same pipettes for each run using 2 + 18ul dilutions, I am keeping everything consistent in regards to my technique and equipment.
Try taking an aliquot of your standard and aliquotting that into new low-bind tubes that are stored at 4 degrees instead. I assume your pipettes are recently calibrated? Perhaps try 45ul and 5ul standard? You could also add carrier DNA (e.g. 10-100ng/ul tRNA) to your diluent.
Is this a new qPCR assay? Or a published one?
Jessica, it depends how far you dilute the International Standard (IS). If you use 1:1 and 1:10 it should be able to give you more than 90% efficiency unless you have issues with your reagents. Are you trying to measure parasite level using absolute quantification?
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