Daniel:Notebook/ComboLock/2017-4-11

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RCA Double Deuce[edit]

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Experiment is going to test how well RCA works (again). For this experiment I will be using 2 primers, the LLRC***, which targets the + strand (that is the padlock/circular amplicon itself) and primer12RC, which targets the (-) strand. This should create extra amplification and

Protocol[edit]

  1. Rolling Circle Amplification
    1. Prepare master mix according to table below
    2. Reagent Single Rxn (uL) Master Mix X+ (3.2X) Master Mix X- (3.2X) Master Mix Y+ (3.2X) Master Mix Y- (3.2X) Joker
      Enzyme NA NEB NEB Thermo Thermo NEB
      Circular Product (1X-1nM) 2 6.4 0 6.4 0 2
      Linear Product (1X) 2 0 6.4 0 6.4 0
      RCA Primer (1 uM each) 2 0 0 0 0 2***
      dNTP (1 mM) 5 16 16 16 16 5
      10X Buffer 2 6.4 6.4 6.4 6.4 2
      Phi29 1 0 0 0 0 1
      BSA (10 mg/mL) 0.4 1.28 1.28 0 0 0.4
      nfH2O 5.6 17.92 17.92 19.2 19.2 7.6
      Total 20 48 48 48 48 20
    3. Add 15 uL master mix to each tube
      1. Samples "A-C" get + master mixes; samples "D-F" get - master mixes
    4. Add 1 uM RCA primer and to new reaction tubes according to following rules
      1. "A,D" samples get 2 uL each LLRC & primer12RC
      2. "B,E" samples get 2 uL each LLRC*** & primer12RC
      3. "C,F" samples get 4 uL LLRC***
    5. Heat reactions up to 95C for 5 min
    6. Cool to 55C and incubate for 15 minutes
    7. Cool to 30C and add enzyme
    8. Incubate for 3 hours at 30C
    9. Incubate for 65 minutes at 20C to denature

Continued tomorrow