Daniel:Notebook/ComboLock/2016-10-4
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Primer24 qPCR Test (From Yesterday)[edit]
Continuing the primer amplifications to test off-target binding.
Protocol[edit]
- qPCR
- Set up qPCR reactions according to table
- Make the following master mix
- 90.2 uL nfH2O
- 2.05 uL 100 uM primer12 RC
- 2.05 uL 10 uM Amp6.3R Index 30
- 102.5 uL Kapa SYBR Fast
- Aliquot 43 uL MM1 into each well
- Add 2 uL sample to appropriate well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 48 uL TBE, 12 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Molecule Count | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM primer24 | 100 uM AmpF | H2O | Total Volume (uL) |
Sample 0A | 1.00E+09 | A1 | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 0B | 1.00E+09 | A2 | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 0C | 1.00E+09 | A3 | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
NTC (rev) | 0 | A4 | 0 | 25 | 0.5 | 0.5 | 22 | 50 |
Results[edit]
- 2016-10-04-qPCR-LatchX4-Primer24-AmpF.png
qPCR Curve
- 2016-10-04-LatchX4-AmpF-Primer24.png
Gel image