Daniel:Notebook/ComboLock/2016-7-29

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Revision as of 19:26, 29 July 2016 by >Djacobse (→‎Protocol)
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Phusion Test (Started yesterday)

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Protocol

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
    2. Chill on ice 1 min
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 1 uL exonuclease I (20 units/μL) and 1 uL exonuclease III (100 units/μL) per sample
    2. Reagent Stock Concentration Reagent Added uL Added per sample Master Mix (5.1X)
      ExoI 20U/uL 20 1 5.1
      ExoIII 100 U/uL 100 1 5.1
    3. Add 2 μL of exonuclease I/III mix to reaction
    4. Mix the reaction by swirling pipette around the well 5 times
    5. Incubate reaction at 37 ºC for 2 hours
    6. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR
    1. Set up reaction according to table
    2. Sample Lane AmpR Index Cells 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 (Hs-Hemo Klentaq) A1 15 2.5 25 0.5 5 17 50
      Sample 2 (Hs-Phusion) B1 16 2.5 25 0.5 5 17 50
      Sample 3 (Hs-No C-Probe) C1 17 2.5 25 0.5 5 17 50
      Sample 4 (mm-Phusion) D1 18 2.5 25 0.5 5 17 50
      Sample 5 (NTC) E1 19 2.5 25 0.5 5 17 50
      Sample 6 (No Sample) F1 20 2.5 25 0.5 5 19 50
    3. Master Mix
      1. 152.5 uL KAPA SyberFast MM
      2. 103.7 uL nfH2O
      3. 3.05 uL AmpF 6.4 100 uM
    4. Add 43 uL Master Mix to each well
    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 x20
      7. 72C 2 min
      8. 16C hold

    qPCR Results