Daniel:Notebook/ComboLock/2016-8-23

From ZhangLabWiki
Revision as of 16:45, 23 August 2016 by >Djacobse (→‎Protocol)
Jump to navigation Jump to search

Positive Control Amplicon Test 2 (Started yesterday)

Back to Calendar

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 Name 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 1 X2-1X A1 33 2 25 0.5 5 17.5 50
      Sample 2 X2-5X B1 34 2 25 0.5 5 17.5 50
      Sample 3 X2-10X C1 35 2 25 0.5 5 17.5 50
      Sample 4 X2-50X D1 36 2 25 0.5 5 17.5 50
      Sample 5 X3-1X E1 37 2 25 0.5 5 17.5 50
      Sample 6 X3-5X F1 38 2 25 0.5 5 17.5 50
      Sample 7 X3-10X G1 39 2 25 0.5 5 17.5 50
      Sample 8 X3-50X H1 40 2 25 0.5 5 17.5 50
      Sample 9 X2Flip A12 41 2 25 0.5 5 17.5 50
      Sample 10 X3Flip B12 42 2 25 0.5 5 17.5 50
      Sample 9 PCR NTC C12 43 2 25 0.5 5 19.5 50
    3. Master Mix
      1. 277.5 uL KAPA SyberFast MM
      2. 194.25 uL nfH2O
      3. 5.55 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