Daniel:Notebook/ComboLock/2016-9-23

From ZhangLabWiki
Revision as of 18:01, 23 September 2016 by >Djacobse (→‎Control)
Jump to navigation Jump to search

PCR Testing (Started Yesterday

Back to Calendar

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

Control

Standard qPCR like I normally do. Control version for the tests after.

  1. 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 1A 1.00E+10 A1 34 2 25 0.5 5 17.5 50
      Sample 1B 1.00E+10 B1 34 2 25 0.5 5 17.5 50
      Sample 1C 1.00E+10 C1 34 2 25 0.5 5 17.5 50
      Sample 2A 1.00E+09 D1 35 2 25 0.5 5 17.5 50
      Sample 2B 1.00E+09 E1 35 2 25 0.5 5 17.5 50
      Sample 2C 1.00E+09 F1 35 2 25 0.5 5 17.5 50
      NTC 0 A1 36 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

    Primer Concentration

    1. qPCR
      1. Set up reaction according to table
      2. Master Mix
        1. 480 uL KAPA SyberFast MM
        2. 336 uL nfH2O
        3. 9.5 uL AmpF 6.4
      3. Add 43 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 x20
        7. 72C 2 min
        8. 16C hold


      Annealing Temperature

      1. qPCR
        1. Set up reaction according to table
        2. Master Mix
          1. 480 uL KAPA SyberFast MM
          2. 336 uL nfH2O
          3. 9.5 uL AmpF 6.4
        3. Add 43 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 x20
          7. 72C 2 min
          8. 16C hold

        Extension Time

        1. qPCR
          1. Set up reaction according to table
          2. Master Mix
            1. 480 uL KAPA SyberFast MM
            2. 336 uL nfH2O
            3. 9.5 uL AmpF 6.4
          3. Add 43 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 x20
            7. 72C 2 min
            8. 16C hold