Daniel:Notebook/ComboLock/2017-6-16

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Antibody Detection Test-Positive Control (Started Wednesday)[edit]

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Protocol-Part 5 (From Yesterday)[edit]

  1. Bglii digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (6.2X)
      RCA Rxn 7 0
      10X Buffer 3.1 2 12.4
      Bglii 1 6.2
      nfH2O 10 62
      Total 20 80.6
    3. Aliquot 12 uL master mix into new tube
    4. Add 7 uL appropriate sample
    5. Mix with vortexer, spin down, and add 1 uL Bglii
    6. Incubate 2 hr 37C
    7. Heat kill with 65C for 20 min
    8. Hold at 12C
  2. qPCR
    1. Make the following 7.2X master mix (1X)
      1. 7.2 uL 10 uM AmpF6.4Sol
      2. 151.2 uL (21) nfH2O
      3. 180 uL 2X Kapa SYBR Fast Master Mix
    2. Add 47 uL master mix to each lane
    3. Add 1 uL AmpR and 2 uL sample according to plate layout
    4. File:PlateLayout-20170616-AntibodyTest.png
    5. Use the following protocol
      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
  3. TBE Gel
    1. Mix together 120 uL TBE and 30 uL 6X dye
    2. Aliquot 10 uL/lane onto parafilm
    3. Add 2 uL correct sample or (1.5 uL for ladder) to aliquots
    4. Add 10 uL aliquots to correct lanes
    5. Run gel at 235V for 24 minutes
    6. Remove gel from case and incubate in TBE buffer with 2 uL SYBR Gold for 3 minutes
    7. Image in gel doc

Results[edit]