Daniel:Notebook/ComboLock/2016-9-23

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Revision as of 17:36, 26 September 2016 by >Djacobse (→‎Extension Time)
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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. Make master mixes according to following table
    4.   Master Mix
      Reagent MMX MMO NTC
      PrimerF (100 uM) 9.05 1.81 3.05
      PrimerR (10 uM) 90.5 18.1 30.5
      KAPA Master Mix 452.5 452.5 152.5
      nfH2O 316.75 396.39 118.95
    5. Add 48 uL correct master mix to each well
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold

    qPCR Sample Table

    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) Master Mix
    Sample 1A 1.00E+10 A1 34 2 25 0.5 5 17.5 50 MMX
    Sample 1B 1.00E+10 B1 34 2 25 0.5 5 17.5 50 MMX
    Sample 1C 1.00E+10 C1 34 2 25 0.5 5 17.5 50 MMX
    Sample 1A 1.00E+09 D1 35 2 25 0.1 1 21.9 50 MMO
    Sample 1B 1.00E+09 E1 35 2 25 0.1 1 21.9 50 MMO
    Sample 1C 1.00E+09 F1 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 H1 36 0 25 0.5 5 19.5 50 NTC
    Sample 1A 1.00E+10 A3 34 2 25 0.5 5 17.5 50 MMX
    Sample 1B 1.00E+10 B3 34 2 25 0.5 5 17.5 50 MMX
    Sample 1C 1.00E+10 C3 34 2 25 0.5 5 17.5 50 MMX
    Sample 1A 1.00E+09 D3 35 2 25 0.1 1 21.9 50 MMO
    Sample 1B 1.00E+09 E3 35 2 25 0.1 1 21.9 50 MMO
    Sample 1C 1.00E+09 F3 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 H3 36 0 25 0.5 5 19.5 50 NTC
    Sample 1A 1.00E+10 A5 34 2 25 0.5 5 17.5 50 MMX
    Sample 1B 1.00E+10 B5 34 2 25 0.5 5 17.5 50 MMX
    Sample 1C 1.00E+10 C5 34 2 25 0.5 5 17.5 50 MMX
    Sample 1A 1.00E+09 D5 35 2 25 0.1 1 21.9 50 MMO
    Sample 1B 1.00E+09 E5 35 2 25 0.1 1 21.9 50 MMO
    Sample 1C 1.00E+09 F5 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 H5 36 0 25 0.5 5 19.5 50 NTC
    Sample 2A 1.00E+10 A7 34 2 25 0.5 5 17.5 50 MMX
    Sample 2B 1.00E+10 B7 34 2 25 0.5 5 17.5 50 MMX
    Sample 2C 1.00E+10 C7 34 2 25 0.5 5 17.5 50 MMX
    Sample 2A 1.00E+09 D7 35 2 25 0.1 1 21.9 50 MMO
    Sample 2B 1.00E+09 E7 35 2 25 0.1 1 21.9 50 MMO
    Sample 2C 1.00E+09 F7 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 H7 36 0 25 0.5 5 19.5 50 NTC
    Sample 2A 1.00E+10 A9 34 2 25 0.5 5 17.5 50 MMX
    Sample 2B 1.00E+10 B9 34 2 25 0.5 5 17.5 50 MMX
    Sample 2C 1.00E+10 C9 34 2 25 0.5 5 17.5 50 MMX
    Sample 2A 1.00E+09 D9 35 2 25 0.1 1 21.9 50 MMO
    Sample 2B 1.00E+09 E9 35 2 25 0.1 1 21.9 50 MMO
    Sample 2C 1.00E+09 F9 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 H9 36 0 25 0.5 5 19.5 50 NTC
    Sample 2A 1.00E+10 A12 34 2 25 0.5 5 17.5 50 MMX
    Sample 2B 1.00E+10 B12 34 2 25 0.5 5 17.5 50 MMX
    Sample 2C 1.00E+10 C12 34 2 25 0.5 5 17.5 50 MMX
    Sample 2A 1.00E+09 D12 35 2 25 0.1 1 21.9 50 MMO
    Sample 2B 1.00E+09 E12 35 2 25 0.1 1 21.9 50 MMO
    Sample 2C 1.00E+09 F12 35 2 25 0.1 1 21.9 50 MMO
    NTC 0 H12 36 0 25 0.5 5 19.5 50 NTC

    Results

    The qPCR lanes are averages for sample lanes (not for NTCs)

    Discussion

    Well, I'm not sure there is a clear indication, at least at the temperature range I used, that there is a temperature dependence. Comparing the 65C (theoretically the most stringent) and the 50C (least stringent) show little difference overall between the off-target bands. The lowered primer concentration does seem to help though, so I can try again on Monday with a continually decreasing level of primers.