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Initial QPCR Program (Opticon):
Heat:
98°C – 30 seconds
Amplify:
98°C – 30 seconds
52°C – 30 seconds
72°C – 30 seconds
Cool:
4°C - continuous
Use Ct (bottom of curve, not mid-log) of curves to determine dilutions for step 1 amplification (Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls)
Breakdown of QPCR amplification math (done to normalize each sample):
○ delta Ct = Sample Ct - lowest highest Ct in sample set (lowest concentration, note: must be Ct of 20 or below)
○ fold = 1.75^(delta Ct)
○ dilution needed = fold
○ note - that input is 2uL per RXN so sample with lowest Ct gets 2uL undiluted
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
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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 highest Ct in sample set (lowest concentration)
○ 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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