Daniel:Notebook/ComboLock/2017-6-7
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Sensitivity and Specificity Experiments (Started Monday)[edit]
Protocol[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-SensSpec-20170607.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 together 120 uL TBE and 30 uL 6X dye
- Aliquot 10 uL/lane onto parafilm
- Add 2 uL correct sample or (1.5 uL for ladder) to aliquots
- Add 10 uL aliquots to correct lanes
- Run gel at 235V for 24 minutes
- Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes
- Image in gel doc
Results[edit]
- PlateLayout-SensSpec-20170607.png
Plate Layout
- SensSpec-PostBglii-20170607-plateCTs.png
Raw CT values
- SensSpec-PostBglii-20170607-plateCTs-normed.png
CT values normalized to NTC (Lane H3)
- SensSpec-PostBglii-20170607-rawcurves.png
Raw curves
- 2017-06-07-SensSpec-PostBglii.png
Gel Image