Daniel:Notebook/ComboLock/2017-1-24

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Revision as of 17:27, 24 January 2017 by >Djacobse (Created page with "=Circularization Test (Started Daniel:Notebook/ComboLock/2017-1-23|Yesterday]])= Back to Calendar= ==Protocol-Part 2== <ol start="3"> <li>Exon...")
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Circularization Test (Started Daniel:Notebook/ComboLock/2017-1-23|Yesterday]])

Back to Calendar=

Protocol-Part 2

  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 1.5 hours at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare 7X Master mix according to table below
    2. Add 15 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (8.5X)
      Template 5 NA
      RCA Primer (10 uM) 2.5 21.25
      dNTP (1 mM) 0.8 6.8
      10X Buffer 2 17
      Phi29 1 8.5
      nfH2O 8.7 73.95
      Total 20 127.5
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step