Daniel:Notebook/PosSequencing/2017-6-29

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emPCR 7-Strand Separation (Started Yesterday)

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Protocol (From Yesterday)

  1. Strand Separation (DNA Denaturation)
    1. Split each sample (1 pM and 100 pM) into 4 parts->
    2. Add 500 uL KOH buffer (pH 12.0)
    3. Spin down sample at 13,000rpm for 1 minute
    4. Remove all but 40uL buffer
    5. Repeat KOH steps A-C once more
    6. Add 500 uL 4X SSC buffer (pH 12.0)
    7. Spin down sample at 13,000rpm for 1 minute
    8. Remove all but 10uL buffer
    9. Resuspend in 80 uL 2X SSC buffer
  2. Single primer qPCR
    1. Make the following master mixes
    2. Reagent uL in Single Rxn Master Mix P2 (4.2X) Master Mix BioU (4.2X) Master Mix Dual (6.2X)
      Primer 2 (10 uM) 1 4.2 0 6.2
      BioU Primer (10 uM) 1 0 4.2 6.2
      2X Kapa SYBR Master Mix 20 84 84 124
      nfH2O 16 67.2 67.2 93
      Total* (+ 5 uL sample=40) 35 155.4 155.4 229.4
    3. Aliquot 35 uL master mix to appropriate lanes
    4. Add 2 uL sample according to plate layout below
    5. File:PlateLayout-Beadless-emPCR-singleprimer-20170626.png
    6. Run the following thermocycler program
      1. 95C 3 min
      2. 95C 3 sec
      3. 45C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x90
      7. 72C 2 min
      8. 16C hold