Daniel:Notebook/ComboLock/2017-3-2

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Revision as of 17:30, 2 March 2017 by >Djacobse (→‎Protocol-Part 4)
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Padlock0601 Test (Started Yesterday)

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Protocol-Part 3

  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 1.5 hours at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Column Purification-Qiaquick
    1. Add 150 uL (5X) PB (binding buffer) to 30 uL 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. Repeat wash step;
    5. Dry spin column for 1 minute at 14000 rpm; discard flow through
    6. Let stand with cover open in fume hood for ~5 minutes
    7. Transfer column to a new 1.5 mL eppendorf tube
    8. Elute with 30 uL EB
    9. Let stand 1 minute
    10. Spin for 1 minute at 14000 rpm
  3. Rolling Circle Amplification
    1. Samples will be aliquoted under following rules
    2. Samples Condition
      Sample X (1-4, AB) Unpurified
      Sample Y (1-4, AB) Purified
      A Samples (1-4) Normal Primer
      B Samples (1-4) Phosphorothiorate Primer
    3. Prepare 8.2X Master mix according to table below
    4. Reagent Single Rxn Vol 16.2X MM uL
      Template 4 0
      RCA Primer (10 uM) 2.5 0
      dNTP (1 mM) 5 81
      10X Buffer 2 32.4
      Phi29 1 0
      BSA (10 mg/mL) 0.4 6.48
      nfH2O 5.1 82.62
      Total 20 202.5
    5. Add 12.5 uL master mix to each tube
    6. Add 4 uL template according to table
    7. Add 2.5 uL correct primer according to table
    8. Incubate at 95 C for 5 minutes
    9. Lower to 55C for 15 minutes
    10. Lower to 30C for at least 2 minutes
    11. Add 1 uL Phi29 polymerase to each sample
    12. Incubate 3 hours at 30C
    13. Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C

Protocol-Part 4

  1. BglII Digestion
  2. qPCR