Daniel:Notebook/ComboLock/2017-4-29

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Revision as of 17:18, 29 April 2017 by >Djacobse (Created page with "=Production Run (Started Yesterday)= Back to Calendar ==Protocol-Part 4== <ol start="7"> <li>Exonuclea...")
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Production Run (Started Yesterday)

Back to Calendar

Protocol-Part 4

  1. Exonuclease Digestion
    1. Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1.5 hours at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare master mix according to table below
    2. Reagent Single Rxn (uL) Master Mix X (10.2X) Master Mix Y (6.2X)
      Enzyme NA NEB Thermo
      Sample 4 0 0
      RCA Primer (1 uM each) 2 20.4 12.4
      dNTP (1 mM) 5 51 31
      10X Buffer 2 20.4 12.4
      Phi29 1 10.2 6.2
      BSA (10 mg/mL) 0.4 4.08 0
      nfH2O 5.6 57.12 37.2
      Total 20 163.2 99.2
    3. Add 15 uL master mix to each tube
      1. Samples "A-C" get + master mixes; samples "D-F" get - master mixes
    4. Add 1 uM RCA primer and to new reaction tubes according to following rules
      1. "A,D" samples get 2 uL each LLRC & primer12RC
      2. "B,E" samples get 2 uL each LLRC*** & primer12RC
      3. "C,F" samples get 4 uL LLRC***
    5. Heat reactions up to 95C for 5 min
    6. Cool to 55C and incubate for 15 minutes
    7. Cool to 30C and add enzyme
    8. Incubate for 3 hours at 30C
    9. Incubate for 65 minutes at 20C to denature
  • qPCR
    1. Make qPCR master mixes according to following recipe