Daniel:Notebook/ComboLock/2017-2-28

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Revision as of 16:40, 28 February 2017 by >Djacobse (→‎Protocol)
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RCA + Circularization Test (Started Yesterday)

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Protocol

  1. ssQubit
    1. Mix together 796 uL nfH2O and 4 uL dye (200:1 ratio)
    2. Add 190 uL mix and 10 uL S1 or S2 to standard tubes
    3. Add 199 uL mix and 1 uL sample to sample tubes
    4. Measure in Qubit; use new run and calibrate with standards
  2. Column Purification-Qiaquick
    1. Add 5X PB (binding buffer) to sample
    2. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    3. Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    4. Dry spin column for 1 minute at 14000 rpm; discard flow through
    5. Let stand with cover open in fume hood for ~5 minutes
    6. Transfer column to a new 1.5 mL eppendorf tube
    7. Add ≥30 uL EB to column
    8. Let stand 1 minute
    9. Spin for 1 minute at 14000 rpm
  3. Rolling Circle Amplification
    1. Prepare master mixes with following enzyme layout
    2. Samples Condition
      Sample X (1-2,ABC) NEB
      Sample Y (1-2,ABC) Thermo Fisher
      Sample Z (1-2,ABC) Epicenter
      A Samples (1-2, XYZ) Normal Primer
      B Samples (1-2, XYZ) Phosphorothiorate Primer
      C Samples (1-2, XYZ) Column Purified Product
    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