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Reagent | X1 RXN (uL) | X220 RXN | X431 |
H2O | 12.1 | 2,662 | 5,215.1 |
HF Buffer | 5 | 1,100 | 2,155 |
dNTPs | 0.5 | 110 | 215.5 |
PE16s_V4_U515_F -OR- PE16s_v4_F_Bar## (3uM) | 2.5 | 550 | 1,077.5 |
PE16S_V4_E786_R (3uM) | 2.5 | 550 | 1,077.5 |
Template | 2 | - | - |
SYBR green (1/100 dilu) | 0.125 | 27.5 | 53.9 |
Phusion | 0.25 | 55 | 107.8 |
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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 (google docs, Please reference Illumina Library QPCR and Multiplexing document)
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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