Daniel:Notebook/ComboLock/2017-6-6

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Sensitivity and Specificity Experiments (Started Yesterday

Back to Calendar

Protocol-Part 4

  1. Exonuclease Digestion
    1. Mix 17.5 uL Exo I (20U/uL) and 3.5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 2 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)
      Enzyme NA Thermo
      Sample 4 0
      p12RC*** (10 uM) 0.2 2.04
      RCA Primer LLRC*** (10 uM) 0.2 2.04
      dNTP (10 mM) 0.5 5.1
      10X Buffer 2 20.4
      Phi29 1 0
      BSA (10 mg/mL) 0.4 4.08
      nfH2O 11.7 119.34
      Total 20 153
    3. Add 15 uL master mix to each tube
    4. Add 4 uL sample to appropriate tubes
    5. Heat reactions up to 95C for 5 min
    6. Cool to 55C and incubate for 15 minutes
    7. Cool to 30C and add 1 uL enzyme
    8. Incubate for 3 hours at 30C
    9. Incubate for 65 minutes at 20C to denature
  3. Bglii digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (10.2X)
      RCA Rxn 7 0
      10X Buffer 3.1 2 20.4
      Bglii 1 0
      nfH2O 10 102
      Total 20 122.4
    3. Aliquot 12 uL master mix into new tube
    4. Add 7 uL appropriate sample
    5. Mix with vortexer, spin down, and add 1 uL Bglii
    6. Incubate 2 hr 37C
    7. Heat kill with 65C for 20 min
    8. Hold at 12C