Daniel:Notebook/ComboLock/2016-8-28

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Revision as of 16:38, 29 August 2016 by >Djacobse (→‎Protocol)
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Positive Control Dilution Test (Started Friday)

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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 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
  3. qPCR
    1. Set up reaction according to table
    2. Sample Molecule Count Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 0 1.00E+11 A1 34 2 25 0.5 5 17.5 50
      Sample 1 1.00E+10 B1 35 2 25 0.5 5 17.5 50
      Sample 2 1.00E+09 C1 36 2 25 0.5 5 17.5 50
      Sample 3 1.00E+08 D1 37 2 25 0.5 5 17.5 50
      Sample 4 1.00E+07 E1 38 2 25 0.5 5 17.5 50
      Sample 5 1.00E+06 F1 39 2 25 0.5 5 17.5 50
      Sample 6 100000 G1 41 2 25 0.5 5 17.5 50
      Sample 7 10000 H1 42 2 25 0.5 5 17.5 50
      Sample 8 1000 A12 43 2 25 0.5 5 17.5 50
      Sample 9 100 B12 44 2 25 0.5 5 17.5 50
      Sample 10 10 C12 45 2 25 0.5 5 17.5 50
      Sample 11 1 D12 46 2 25 0.5 5 17.5 50
      Sample 12 0 E12 47 2 25 0.5 5 19.5 50
    3. Master Mix
      1. 327.5 uL KAPA SyberFast MM
      2. 229.25 uL nfH2O
      3. 6.55 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
  4. qPCR Standard
    1. Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC
    2. Master Mix
      1. 352.5 uL KAPA SyberFast MM
      2. 310.2 uL nfH2O
      3. 7.05 uL primer2
      4. 7.05 uL primer12RC
    3. Add 48 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 x25
      7. 72C 2 min
      8. 16C hold

      Results