Daniel:Notebook/ComboLock/2017-6-7

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Sensitivity and Specificity Experiments (Started Monday)[edit]

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Protocol[edit]

  1. qPCR
    1. Make 17.2X master mix according to following (1X)
      1. 361.2 uL nfH2O (21)
      2. 17.2 uL 10 uM AmpF6.4Sol (1)
      3. 430 uL 2X Kapa SYBR Fast Master Mix (25)
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL AmpR Index (10 uM) according to plate layout
    4. File:PlateLayout-SensSpec-20170607.png
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
  3. Mix together 120 uL TBE and 30 uL 6X dye
  4. Aliquot 10 uL/lane onto parafilm
  5. Add 2 uL correct sample or (1.5 uL for ladder) to aliquots
  6. Add 10 uL aliquots to correct lanes
  7. Run gel at 235V for 24 minutes
  8. Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes
  9. Image in gel doc

Results[edit]