Daniel:Notebook/ComboLock/2016-7-19

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C Probe Test 3-SOD1/VIM-Stringent Washes (Started yesterday)[edit]

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Protocol[edit]

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 10 uL exonuclease I (20 units/μL) and 2 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 Lane AmpR Index Cells 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1A A1 21 2 25 0.5 5 17.5 50
      Sample 2A B1 22 2 25 0.5 5 17.5 50
      Sample 3A C1 23 2 25 0.5 5 17.5 50
      Sample 4A D1 24 2 25 0.5 5 17.5 50
      Sample 5A E1 25 2 25 0.5 5 17.5 50
      Sample 1B F1 26 2 25 0.5 5 17.5 50
      Sample 2B G1 27 2 25 0.5 5 17.5 50
      Sample 3B H1 28 2 25 0.5 5 17.5 50
      Sample 4B A12 29 2 25 0.5 5 17.5 50
      Sample 5B B12 30 2 25 0.5 5 17.5 50
      Sample 6 C12 31 0 25 0.5 5 19.5 50
    3. Master Mix
      1. 302.5 uL KAPA SyberFast MM
      2. 192.5 uL nfH2O
      3. 6.05 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

    qPCR Results[edit]