Hello everyone,
For the quantification of a product (mix of 2 plasmids) by qPCR/ddPCR, we obtained inconsistent results between the different 10 fold dilutions in nuclease-free water (dilution series: 10 µL plasmids + 90 µL nuclease-free water). The same assay has been performed several times and the results are always inconsistent (different days, different technicians...).
For the reference standard, the efficiency is good (between 90 and 110%), but the delta Ct varies between the dilutions (for example: 2.3 Ct, then 3.3 Cp...)
The following technical points have been optimized: annealing temperature, mastermix, primers/probes.
Do you have any explanations or suggestions ?
Thank you in advance,
Florian
Are the plasmids circular or did you digest them?
Sometimes you can get secondary structure in circular plasmids that makes amplification difficult or inefficient.
Try using a single-cutter, make certain it does NOT cut in your region inside your PCR product(s), and see if that makes a difference.
Variation can also be due to repeated freeze-thaws of the reagents. Most folks will aliquot their standards into "single experiment size" aliquots to thaw once, use once, and toss any extras.
Good luck!
Dear Katie, thank you for your answer.
Yes, the plasmids are in the circular form.
We performed this test yesterday: digestion with a restriction enzyme that does not cut in the target gene. We are performing the qPCR today.
Regarding the repetition of freeze-thaws of the reagents, we use aliquots for the primers/probes and the mastermix is stored at 2-8°C.
Florian
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