Daniel:Notebook/PosSequencing/2017-6-5
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Aqueous PCR Test (Started Friday June 2)[edit]
Given the results from Saturday I'm going to repeat qPCR for the E fraction after baking for 15 minutes.
Protocol[edit]
- Baking
- Incubate fraction "E" for 15 minutes at 95C
- Chill on ice prior to adding to qPCR lanes
- qPCR
- Make the following 6.2X master mix (1X)
- 6.2 uL 10 uM BiotinUracilPrimer1 (1)
- 6.2 uL 10 uM Primer2 (1)
- 124 uL 2X Kap SYBR Master Mix (20)
- 99.2 uL nfH2O (16)
- Aliquot 38 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-20170605-aqPCR-EFraction.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x120
- 72C 2 min
- 16C hold
Results[edit]
- PlateLayout-20170605-aqPCR-EFraction.png
Plate layout
- 20170605-aqPCR-EFraction-plateCTs.png
Plate CT values
- 20170605-aqPCR-EFraction-plateCTs-normed.png
Plate CT values-normalized
- 20170605-aqPCR-EFraction-rawcurves.png
Raw curves