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Materials used:
○ Contents of MM
○ P200 multi-channel pipette
○ 96 well QPCR plate (96 well for opticon stocked in lab)
○ Clear QPCR plate covers Initial QPCR master mix (MM)| Reagent |
Reagent | X1 RXN (uL) |
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H2O | 12.1 |
HF Buffer | 5 |
dNTPs | 0.5 |
PE16s_V4_U515_F (3uM) | 2.5 |
PE16S_V4_E786_R (3uM) | 2.5 |
Template | 2 |
SYBR green (1/100 dilu) | 0.125 |
Phusion | 0.25 |
Run this step in duplicate or triplicate to best estimate proper cycling time
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 (google docs, Illumina Library QPCR and Multiplexing)
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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1st step Master Mix 25uL RXN (MM1)
Reagent | X1 RXN (uL) |
H2O | 12.25 |
HF Buffer | 5 |
dNTP | 0.5 |
PE16S_V4_U515_F F (3uM) | 2.5 |
PE16S_V4_E786_R (3uM) | 2.5 |
Template | 2 |
Phusion | 0.25 |
16s Step 16SStep 1 Program:
Heat:
98°C – 30 seconds
Amplify:
98°C – 30 seconds
52°C – 30 seconds
72°C – 30 seconds
Cool:
4°C - continuous
Run amplification cycle number determined via QPCR (no more than 20 cycles allowed)
After cycling pool duplicates, now have 1x 100uL reaction per sample
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Sample Re-Aliquoting and Step 2
Please Note: Samples are run as four 25uL reactions that are pooled at end of cycling 2nd step Master Mix 25uL RXNs (MM2)| Reagents |
Reagents | X1 RXN (uL) |
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H2O | 8.65 |
HF Buffer | 5 |
dNTPs | 0.5 |
PE-PCR-III-F (3uM) | 3.3 |
PE-PCR-IV-XXX (3uM) | 3.3 |
Template | 4 |
Phusion | 0.25 |
16s Step 2 Program:
Heat:
98°C – 30 seconds
Amplify:
98°C – 30 seconds
83°C – 30 seconds
72°C – 30 seconds
Cool:
4°C - continuous
Run 7 cycles of amplification
- After cycling pool duplicates, now have 1x 100uL reaction per sample
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Final QPCR
Once you have a substantial or all of your samples prepared you can run a final QPCR to determine dilutions and volumes for multiplexing. This step also confirms that the library preparation was successful QPCR Master Mix (QPCR MM, 20uL RXN)| Reagents | X1 RXN (uL) | X345RXN
Reagents | X1 RXN (uL) |
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H2O | 7.22 ,484 | |
PE Seq Primer – F (10uM) | 0.4 138 | |
PE Seq Primer – R (10uM) | 0.4 | 138 |
KAPA SYBRgreen MM | 10 | 3,450 |
Template | 2 - |
Final QPCR Program (Opticon or Lichtcycler)
Heat:
95°C – 5 minutes
Amplify:
95°C – 10 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 seconds
Run 35 cycles of amplification
Use mid-log phase of curves to determine volumes for multiplexing (google docs, Illumina Library QPCR and Multiplexing)
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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