Daniel:Notebook/ComboLock/2017-2-3

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Revision as of 17:22, 3 February 2017 by >Djacobse (→‎Protocol-Part 4-RCA)
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Version 2 Oligos Round 2 (Started yesterday)

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Protocol-Part 4-RCA

  1. Exonuclease Digestion
    1. Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1 hour at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare 8.2X Master mix according to table below
    2. Reagent uL Added Master Mix (8.2X)
      Template 5 NA
      RCA Primer (10 uM) 2.5 20.5
      dNTP (1 mM) 5 41
      10X Buffer 2 16.4
      Phi29 1 0
      BSA (10 mg/mL) 0.4 3.28
      nfH2O 4.1 33.62
      Total 20 131.2
    3. Add 14 uL master mix to each tube
    4. Add sample to new reaction tubes
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step

Protocol Part 5-qPCR and Gel

  1. qPCR
    1. Make 9.2X qPCR master mix according to following recipe
      1. 193.2 uL nfH2O
      2. 230 uL SYBR Fast
      3. 9.2 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section