Daniel:Notebook/PosSequencing/2017-6-3: Difference between revisions
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==Results== | |||
<gallery perrow=2 heights=300px widths=400px mode=packed-hover> | |||
File:PlateLayout-20170603-aqPCR.png|Plate layout | |||
File:20170603-aqPCR-Fractions-plateCTs.png|Plate CT values | |||
File:20170603-aqPCR-Fractions-norm-plateCTs.png|Plate CT values-normalized | |||
File:20170603-aqPCR-Fractions-rawcurves.png|Raw curves | |||
</gallery> |
Revision as of 23:19, 3 June 2017
Aqueous PCR Test (Started Yesterday)
Protocol
- Wash Steps
- Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: A)
- Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
- Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: B)
- Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
- Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: C)
- Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
- Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: D)
- Add 50 uL 4X SSC buffer to wash, pipette up and down to mix; Keep this fraction (Fraction: E)
- qPCR
- Make the following 14.2X master mix (1X)
- 14.2 uL 10 uM BiotinUracilPrimer1 (1)
- 14.2 uL 10 uM Primer2 (1)
- 284 uL 2X Kap SYBR Master Mix (20)
- 227.2 uL nfH2O (16)
- Aliquot 38 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-20170603-aqPCR.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
- PlateLayout-20170603-aqPCR.png
Plate layout
- 20170603-aqPCR-Fractions-plateCTs.png
Plate CT values
- 20170603-aqPCR-Fractions-norm-plateCTs.png
Plate CT values-normalized
- 20170603-aqPCR-Fractions-rawcurves.png
Raw curves