Below are the Component that I used in my ddPCR reaction
Water 5uL
MasterMix 12.5uL
20x FAM 1.25uL
20x VIC 1.25uL
digested DNA 6.6 ng/uL 5uL
No Call generally means there was no classification possible (either positive or negative result). The software for whatever reason cannot discriminate between positive and negative results. The question you have to answer is why.
ABS plate analysis determines concentrations whether using automatic threshold is set. QuantaSoft will return a No Call for (a) wells with too many positive droplets (not enough empty droplets for Poisson statistics), (b) for wells with Quality Scores below 0.85, and (c) wells with less than 10,000 droplets.
If everything is alright, you may set a threshold manually and you will get calculation.
Could be due to too high concentration of the target, or more optimization needed fr your experiment. Check also the quality of your DNA extraction - I see digested DNA in ng there. How many copies? should be not exceeding 5,000 per reaction. Do you have a quantification curve to refer to? To have a Call you run ABS experiment type, right?
"QuantaSoft software will return a No Call for wells with too many positive droplets
(not enough empty droplets to apply Poisson statistics) ". See bulletin 6407
You don't have negative dropltes in ch2
Dear Doinita Ispas,
What do you mean ABS experiment type?
My study on copy number variation. Channel 1: FAM (unknown) ; Channel 2: VIC (Reference)
33ng per microliter in each sample.
Sorry please could you assist me.
Here is the data
Thanks for your earlier feedback.
ABS stands for absolute quantification.
For some CNV evaluation work please study the papers attached. I think that the DNA amount depends on your sample type.
I don't understand the syntagm "33 ng per uL in each sample", do you mean the concentration of your DNA in your samples or your input into the reaction volume?
Thank you so much Doinita Ispas. Here is the pic explaining the 33ng..
I am getting similar results with most of my samples showing NO CALL. Not sure why? I added the primers and probes at the right concentration, 100 ng/ul samples. Any suggestion would be much appreciated.
Human DNA greater than 66ng/20ul reaction negatively affect the accuracy of quantification and need fragmentation by restriction enzymes.
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