Daniel:Notebook/ComboLock/2016-8-19

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Positive Control Amplicon Test (Started yesterday)

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Protocol

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 uL exonuclease III (100 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. Elution
    1. Add 25 uL prewarmed elution buffer; vortex to suspend
    2. Incubate at RT for 2 minutes
    3. Apply magnet and transfer supernatant to a clean centrifuge tube
    4. Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution
  4. qPCR
    1. Set up reaction according to table
    2. Master Mix
      1. 302.5 uL KAPA SyberFast MM
      2. 192.5 uL nfH2O
      3. 6.05 uL AmpF 6.4
    3. Add 43 uL Master Mix to each well
    4. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x20
      7. 72C 2 min
      8. 16C hold