Daniel:Notebook/PosSequencing/2017-6-9
Jump to navigation
Jump to search
Aqueous PCR 2 (Started Tuesday)[edit]
Protocol[edit]
- Supernatant Extraction
- Vortex qPCR lanes from Tuesday for several seconds
- Magnet pulldown for 1 minute; remove supernatant and keep, putting into new 0.2 mL tubes
- qPCR
- Make the following 6.2X (1X) master mix
- 142.6 (23) uL nfH2O
- 6.2 (1) uL 10 uM Biotin-Uracil primer
- 6.2 (1) uL 10 uM Primer 2
- 155 (25) uL Kapa 2X SYBR master mix
- Add 50 uL (resuspend beads) to each of the 6 lanes from Tuesday's qPCR following plate layout File:PlateLayout-20170609-aqPCRStage2.png
- Make the following 8.2X master mix
- 147.6 (18) uL nfH2O
- 8.2 (1) uL 10 uM Biotin-Uracil primer
- 8.2 (1) uL 10 uM Primer 2
- 205 (25) uL Kapa 2X SYBR master mix
- Add 45 uL master mix to each of the H lanes following plate layout
- Add 5 uL appropriate supernatant, 1 pM barcode 2 oligo, or nfH2O following plate layout
- Apply caps tightly, vortex 5 seconds and spin down
- Use following cycles for qPCR
- 95C 2 min
- 95C 20 sec
- 58C 30 sec
- 70C 30 sec
- Goto b 40 times
- 4C forever
Results[edit]
- PlateLayout-20170609-aqPCRStage2.png
Plate Layout
- 20170609-aqPCRStage2-plateCTs.png
Raw CT values
- 20170609-aqPCRStage2-plateCTs-normed.png
CT values normalized to NTC (Lane A6)
- 20170609-aqPCRStage2-rawcurves.png
Raw curves
- 2017-06-09-aqPCR-beadFraction.png
Gel image-bead fraction
- 2017-06-09-aqPCR-Supernatant.png
Gel image-supernatant fraction
- 2017-06-09-aqPCR-beadFraction-BAKE.png
Gel image-bead fraction post 95C bake (12 min)