Daniel:Notebook/PosSequencing/2017-6-9: Difference between revisions
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>Djacobse |
>Djacobse m (→Protocol) |
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<li>Apply caps tightly, vortex 5 seconds and spin down</li> | <li>Apply caps tightly, vortex 5 seconds and spin down</li> | ||
<li>Use following cycles for qPCR</li> | <li>Use following cycles for qPCR</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li>95C 2 min</li> | <li>95C 2 min</li> | ||
Line 48: | Line 47: | ||
<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:20170609-aqPCRStage2-rawcurves.png|Raw curves | ||
File:2017-06-09-aqPCR-beadFraction.png|Gel image-bead fraction | File:2017-06-09-aqPCR-beadFraction.png|Gel image-bead fraction | ||
File:2017-06-09-aqPCR-Supernatant.png|Gel image-supernatant fraction | File:2017-06-09-aqPCR-Supernatant.png|Gel image-supernatant fraction |
Latest revision as of 23:59, 12 June 2017
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)