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Initial QPCR Program (Roche Lighcycler 480):
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 (Please reference Illumina Library QPCR and Multiplexing documentConstruction/Tools/Attachments/16s primer_amp_QPCR sheets.xls)
Breakdown of QPCR amplification math (done to normalize each sample):
○ delta Ct = Sample Ct - lowest Ct in sample set
○ 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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- The above protocols import volume information using the Initial Dilution Sample and H2O pages from CSV files (please see XXX files for this purpose)the Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls saved as two individual CSV files and imported to Rosie.
- please run the H2O protocol first
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** Now you have a choice!***
If your sample plate is a mixture of sample types (stool and saliva for example) or you have reason to suspect something went wrong:
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