Daniel:Notebook/ComboLock/2016-11-30

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Extended Latch Test (Started [[Daniel:Notebook/ComboLock/2016-11-29])

Back to Calendar

Sample Matrix

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No C Probe
Sample 4 (AB) No Kinase

Protocol

  1. Exonuclease Digestion
    1. Combine 15 uL of Exonuclease I (20U/uL) and 3 uL Exonuclease III (100U/uL)
    2. Add 2 uL of mix to each reaction
    3. Incubate for 30 minutes to 2 hours at 37C
    4. Heat kill by incubating for 5 minutes at 94C
  2. Rolling Circle Amplification
    1. Prepare 9X master mix according to table below
    2. Reagent Stock Concentration Final Conc/Amt uL Added in 20 uL Rxn Master Mix (9X)
      Template NA NA 5 45
      RCA Primer 10 uM 2 uM 4 36
      dNTP 1 mM 50 uM 1 9
      10X Buffer 10X 1X 2 18
      Phi29 (NEB) 10 U/uL 10 U 1 9
      nfH2O NA NA 7 63
      Total     20 180
    3. Add 15 uL master mix to each tube
    4. Add 5 uL sample to correct reaction tubes
    5. Incubate at 37C for 3 hours
    6. Heat kill by incubating at 65C for 10 minutes