Daniel:Notebook/ComboLock/2016-9-26

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PCR Testing (Started Thursday)[edit]

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Continuing the tests on qPCR for the positive control amplicon using primer concentrations. This is following some of the conclusions from Friday.

Protocol[edit]

  1. qPCR
    1. Follow reaction map below
    2. Sample Molecule Count Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL) Master Mix
      Sample 1A 1.00E+09 A1 36 2 25 5 5 13 50 MM1
      Sample 1B 1.00E+09 A2 36 2 25 5 5 13 50 MM1
      Sample 1C 1.00E+09 A3 36 2 25 5 5 13 50 MM1
      NTC 0 A4 36 0 25 5 5 13 50 NTC
      Sample 1A 1.00E+09 A5 36 2 25 1 1 21 50 MM2
      Sample 1B 1.00E+09 A6 36 2 25 1 1 21 50 MM2
      Sample 1C 1.00E+09 A7 36 2 25 1 1 21 50 MM2
      NTC 0 A8 36 0 25 1 1 21 50 NTC
      Sample 1A 1.00E+09 H1 36 2 25 0.5 0.5 22 50 MM3
      Sample 1B 1.00E+09 H2 36 2 25 0.5 0.5 22 50 MM3
      Sample 1C 1.00E+09 H3 36 2 25 0.5 0.5 22 50 MM3
      NTC 0 H4 36 0 25 0.5 0.5 22 50 NTC
      Sample 1A 1.00E+09 H5 37 2 25 2* 2* 19 50 MM4
      Sample 1B 1.00E+09 H6 37 2 25 2* 2* 19 50 MM4
      Sample 1C 1.00E+09 H7 37 2 25 2* 2* 19 50 MM4
      NTC 0 H8 37 0 25 2* 2* 19 50 NTC
      Sample 1A 1.00E+09 C1 37 2 25 0.2* 0.2* 22.6 50 MM5
      Sample 1B 1.00E+09 C2 37 2 25 0.2* 0.2* 22.6 50 MM5
      Sample 1C 1.00E+09 C3 37 2 25 0.2* 0.2* 22.6 50 MM5
      NTC 0 C4 37 0 25 0.2* 0.2* 22.6 50 NTC
    3. Make master mixes according to following table
    4. Reagent Master Mix
      MM1 MM2 MM3 MM4 MM5
      PrimerF (10 uM) 15.5 3.1 1.55 6.2* 0.62*
      PrimerR (10 uM) 15.5 3.1 1.55 6.2* 0.62*
      KAPA Master Mix 77.5 77.5 77.5 77.5 77.5
      nfH2O 40.3 65.1 68.2 58.9 70.06
    5. Add 48 uL correct master mix to each well
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 60C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x26
      7. 72C 2 min
      8. 16C hold

    Results[edit]