Daniel:Notebook/PosSequencing/2017-6-26

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Beadless emPCR (Started Thursday 6/22/17)

Back to calendar

Since the results on Friday showed viable product (at least in the 100 nM sample), I'm going to finish up that experiment with strand denaturation and single primer qPCR.

Protocol

  1. Strand Separation (DNA Denaturation)
    1. Add 500 uL KOH buffer (pH 12.0)
    2. Spin down sample at 13,000rpm for 1 minute
    3. Remove all but 40uL buffer
    4. Repeat KOH steps A-C once more
    5. Add 500 uL 4X SSC buffer (pH 12.0)
    6. Spin down sample at 13,000rpm for 1 minute
    7. Remove all but 10uL buffer
    8. Resuspend in 80 uL 2X SSC buffer
  2. Single primer qPCR
    1. Make the following master mixes
    2. Aliquot 35 uL master mix to appropriate lanes
    3. Add 2 uL sample according to plate layout below
    4. File:PlateLayout-Beadless-emPCR-singleprimer-20170626.png
    5. 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