Daniel:Notebook/ComboLock/2016-8-30

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Revision as of 17:46, 30 August 2016 by >Djacobse (→‎Protocol)
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X3 Dilution Test (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 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 0A 1.00E+11 A1 34 2 25 0.5 5 17.5 50
      Sample 0B 1.00E+11 B1 34 2 25 0.5 5 17.5 50
      Sample 0C 1.00E+11 C1 34 2 25 0.5 5 17.5 50
      Sample 1A 1.00E+10 D1 35 2 25 0.5 5 17.5 50
      Sample 1B 1.00E+10 E1 35 2 25 0.5 5 17.5 50
      Sample 1C 1.00E+10 F1 35 2 25 0.5 5 17.5 50
      Sample 2A 1.00E+09 G1 36 2 25 0.5 5 17.5 50
      Sample 2B 1.00E+09 H1 36 2 25 0.5 5 17.5 50
      Sample 2C 1.00E+09 A12 36 2 25 0.5 5 17.5 50
      Sample 3A 1.00E+08 B12 37 2 25 0.5 5 17.5 50
      Sample 3B 1.00E+08 C12 37 2 25 0.5 5 17.5 50
      Sample 3C 1.00E+08 D12 37 2 25 0.5 5 17.5 50
      Sample 4A 1.00E+07 E12 38 2 25 0.5 5 17.5 50
      Sample 4B 1.00E+07 F12 38 2 25 0.5 5 17.5 50
      Sample 4C 1.00E+07 G12 38 2 25 0.5 5 17.5 50
      Sample 5A 1.00E+06 H12 39 2 25 0.5 5 17.5 50
      Sample 5B 1.00E+06 A2 39 2 25 0.5 5 17.5 50
      Sample 5C 1.00E+06 B2 39 2 25 0.5 5 17.5 50
      NTC 0 C2 41 0 25 0.5 5 19.5 50


    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

    Results