Daniel:Notebook/ComboLock/2017-3-2

From ZhangLabWiki
Revision as of 17:19, 2 March 2017 by >Djacobse (→‎Protocol-Part 3)
Jump to navigation Jump to search

Padlock0601 Test (Started Yesterday)

Back to Calendar

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. Add 15 uL master mix to each tube
    5. Add sample to new reaction tubes according to following table