Daniel:Notebook/PosSequencing/2017-6-3

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Revision as of 00:14, 3 June 2017 by >Djacobse (→‎Protocol)
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Aqueous PCR Test (Started Yesterday)

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Protocol

  1. Wash Steps
    1. Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: A)
    2. Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
    3. Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: B)
    4. Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
    5. Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: C)
    6. Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
    7. Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: D)
    8. Add 50 uL 4X SSC buffer to wash, pipette up and down to mix; Keep this fraction (Fraction: E)
  2. qPCR
    1. Make the following 14.2X master mix (1X)
      1. 14.2 uL 10 uM BiotinUracilPrimer1 (1)
      2. 14.2 uL 10 uM Primer2 (1)
      3. 284 uL 2X Kap SYBR Master Mix (20)
      4. 227.2 uL nfH2O (16)
    2. Aliquot 38 uL master mix to appropriate lanes
    3. Add 2 uL sample according to plate layout below
    4. File:PlateLayout-20170603-aqPCR.png
    5. Run the following thermocycler program
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x120
      7. 72C 2 min
      8. 16C hold