Daniel:Notebook/ComboLock/2016-9-23

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Revision as of 19:12, 23 September 2016 by >Djacobse (→‎Control)
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PCR Testing (Started Yesterday

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Protocol (all)

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 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


Standard qPCR like I normally do. Control version for the tests after.

  1. qPCR
    1. Follow reaction map below
      • Note:"X"=5 uL primer per reaction; "O" indicates 1 uL primer
      • Note:"TN" is a range of temperatures (gradient) from 50C (Lane 1) to 65C (Lane 12)
        1 2 3 4 5 6 7 8 9 10 11 12
      A X,T1   X,T2   X,T3   X,T4   X,T5     X,T6
      B X,T1   X,T2   X,T3   X,T4   X,T5     X,T6
      C X,T1   X,T2   X,T3   X,T4   X,T5     X,T6
      D O,T1   O,T2   O,T3   O,T4   O,T5     O,T6
      E O,T1   O,T2   O,T3   O,T4   O,T5     O,T6
      F O,T1   O,T2   O,T3   O,T4   O,T5     O,T6
      G NTC   NTC   NTC   NTC   NTC     NTC
      H                        
    2. Set up reaction according to table in next section
    3. Master Mix
      1. 480 uL KAPA SyberFast MM
      2. 336 uL nfH2O
      3. 9.5 uL AmpF 6.4
    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

    Primer Concentration

    1. qPCR
      1. Set up reaction according to table
      2. Master Mix
        1. 480 uL KAPA SyberFast MM
        2. 336 uL nfH2O
        3. 9.5 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


      Annealing Temperature

      1. qPCR
        1. Set up reaction according to table
        2. Master Mix
          1. 480 uL KAPA SyberFast MM
          2. 336 uL nfH2O
          3. 9.5 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

        Extension Time

        1. qPCR
          1. Set up reaction according to table
          2. Master Mix
            1. 480 uL KAPA SyberFast MM
            2. 336 uL nfH2O
            3. 9.5 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