Daniel:Notebook/PosSequencing/2017-6-3: Difference between revisions
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>Djacobse (Created page with "=Aqueous PCR Test (Started Yesterday)= Back to Calendar ==Protocol== <ol start="3"> <li>Wash St...") |
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<li>Make the following 14.2X master mix (1X)</li> | <li>Make the following 14.2X master mix (1X)</li> | ||
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Revision as of 00:10, 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 [[Image:|522x348px]]
- 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