Daniel:Notebook/ComboLock/2017-2-3

From ZhangLabWiki
Revision as of 17:17, 3 February 2017 by >Djacobse (→‎Protocol-Part 4-RCA)
Jump to navigation Jump to search

Version 2 Oligos Round 2 (Started yesterday)

Back to Calendar

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 7X 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