Daniel:Notebook/ComboLock/2016-10-8

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Revision as of 18:56, 8 October 2016 by >Djacobse (→‎qPCR Sample Table)
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Padlock0301 Test (Started yesterday)

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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 in next section
    2. Make master mixes according to following recipes
      1. 334.25 uL nfH2O
      2. 477.5 uL SYBR Fast
      3. 9.55 uL AmpF
    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 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-CL 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
Sample 1A 1.00E+09 A4 25 2 25 0.5 5 17.5 50
Sample 1B 1.00E+09 A5 25 2 25 0.5 5 17.5 50
Sample 1C 1.00E+09 A6 25 2 25 0.5 5 17.5 50
Sample 2 1.00E+10 A7 26 2 25 0.5 5 17.5 50
Sample 3 1.00E+09 H2 27 2 25 0.5 5 17.5 50
Sample 4A 1.00E+10 H5 28 2 25 0.5 5 17.5 50
Sample 4B 1.00E+10 H6 28 2 25 0.5 5 17.5 50
Sample 4C 1.00E+10 H7 28 2 25 0.5 5 17.5 50
Sample 5A 1.00E+09 H8 29 2 25 0.5 5 17.5 50
Sample 5B 1.00E+09 D1 29 2 25 0.5 5 17.5 50
Sample 5C 1.00E+09 D2 29 2 25 0.5 5 17.5 50
Sample 6A 1.00E+09 H8 30 2 25 0.5 5 17.5 50
Sample 6B 1.00E+09 D1 30 2 25 0.5 5 17.5 50
Sample 6C 1.00E+09 D2 30 2 25 0.5 5 17.5 50
NTC 0 D3 31 0 25 0.5 5 19.5 50