Daniel:Notebook/ComboLock/2017-5-1
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Production Run (Started Friday 4/28)[edit]
Protocol-Part 5[edit]
- qPCR
- Make 17.2X master mix according to following (1X)
- 361.2 uL nfH2O (21)
- 17.2 uL 10 uM AmpF6.4Sol (1)
- 430 uL 2X Kapa SYBR Fast Master Mix (25)
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout File:PlateLayout-20170501-ProductionRun-PostBglii.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 120 uL TBE and 40 uL 6X dye
- Aliquot 10 uL per sample on to parafilm
- Add 2 uL sample or 1.5 uL ladder to aliquots
- Mix and add 10 uL per lane to gel
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc
- 20170501-qpCR-ProductionRun-PostBglii.png
qPCR curves (NEB amplified samples blue; TF amplified samples red)
- 2017-05-01-ProductionRun-PostBglii-NEB-NoAmp.png
NEB Gel
- 2017-05-01-ProductionRun-PostBglii-TF-NoAmp.png
Thermo Fisher RCA
- 2017-05-01-ProductionRun-PostBglii-NEB-PostAmp.png
NEB Gel-Post qPCR
- 2017-05-01-ProductionRun-PostBglii-TF-PostAmp.png
ThermoFisher Gel Post qPCR
Although the gel quality is poor, I do like these results, especially the pre-qPCR bands in the ThermoFisher lanes. I'm going to check the results via sequencing. See tomorrow.