Daniel:Notebook/PosSequencing/2017-6-9: Difference between revisions
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<gallery perrow=2 heights=250px widths=350px mode=packed-hover> | <gallery perrow=2 heights=250px widths=350px mode=packed-hover> | ||
File:PlateLayout-20170609-aqPCRStage2.png|Plate Layout | File:PlateLayout-20170609-aqPCRStage2.png|Plate Layout | ||
File:|Raw CT values | File:20170609-aqPCRStage2-plateCTs.png|Raw CT values | ||
File:|CT values normalized to NTC (Lane A6) | File:20170609-aqPCRStage2-plateCTs-normed.png|CT values normalized to NTC (Lane A6) | ||
File:|Raw curves | File:|Raw curves | ||
File:2017-06-09-aqPCR-beadFraction.png|Gel image-bead fraction | File:2017-06-09-aqPCR-beadFraction.png|Gel image-bead fraction |
Revision as of 21:33, 9 June 2017
Aqueous PCR 2 (Started Tuesday)
Protocol
- 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
- 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)
- 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)