Daniel:Notebook/ComboLock/2017-2-28

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RCA + Circularization Test (Started Yesterday)

Back to Calendar

Protocol

  1. Rolling Circle Amplification
    1. Prepare master mixes with following enzyme layout
    2. Samples Phi29 Source
      Sample X (1-2,ABC) NEB
      Sample Y (1-2,ABC) Thermo Fisher
      Sample Z (1-2,ABC) Epicenter
    3. Add 14 uL master mix to each tube
    4. Add 5 uL sample to new reaction tubes according to following table
    5. Reagent Single Rxn Vol MM-X (NEB) uL MM-Y (TF) uL MM-Z (Epi) uL
      Template 5 0 0 0
      RCA Primer (10 uM) 2.5 15.25 15.25 15.25
      dNTP (1 mM) 5 30.5 30.5 30.5
      10X Buffer 2 12.2 12.2 12.2
      Phi29 1 0 0 0
      BSA (10 mg/mL) 0.4 2.44 0 0
      nfH2O 4.1 25.01 27.45 27.45
      Total 20 85.4 85.4 85.4
    6. Incubate for 30 minutes at 75C
    7. Lower temperature to 30C
    8. Add 1 uL Phi29 polymerase and incubate 3 hours at 30C
    9. Heat kill enzyme with 10 minutes at 65C
    10. Hold at 4C overnight