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Final QPCR Program (Opticon or Lichtcycler)
HeatActivation:
95°C – - 5 minutes
Amplify Amplification:
95°C – 10 30 seconds
60°C – 20 seconds
72°C – 30 seconds
Melting Curve:
95°C – 5 seconds
65°C – 1 minute
97°C - continuous
Cool:
40°C – 10 seconds45 seconds (1-step annealing/extension)
Run 35 cycles of amplification
Use mid-log phase of curves to determine volumes for multiplexing (Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls)
Please note – samples may fail due to too little, too much material, or a poor reaction. It is recommended that failed samples be re-run before moving forward
Breakdown of QPCR multiplexing math (done to normalize each sample):
○ delta Ct = Sample Ct - lowest Ct in sample set
○ fold = 1.75^(delta Ct)
○ ratio = 1/fold
○ volume to mix b/c of ratio = X*ratio (X = minimum desired volume per sample)
○ how to dilute = fold
○ note - sample with lowest Ct will get an undiluted Xuls added to final multiplex, X can be raised or lowered to accommodate the needed volume of other samples
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