Daniel:Notebook/ComboLock/2017-3-2

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Padlock0601 Test (Started Yesterday)[edit]

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Protocol-Part 3[edit]

  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[edit]

  1. BglII Digestion
    1. Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
    2. Make the following 4.2X master mix
    3. Reagent Single Rxn Vol 8.2X MM uL
      RCA Reaction Product 5 0
      Bglii Cutter Primer (10 uM) 2 16.4
      10X Buffer 3.1 2 16.4
      Bglii 1 0
      nfH2O 10 82
      Total 20 114.8
    4. Aliquot 14 uL master mix into new tubes
    5. Add 5 uL appropriate sample to each tube
    6. Use the following thermocycler program
      1. 5 min 95C
      2. 10 min 50C
      3. 1 hr 37C; When this cycle starts add 1 uL Bglii
      4. Heat kill with 65C for 20 min
  2. qPCR
    1. Make 25.2X qPCR master mix according to following recipe
      1. 529.2 uL nfH2O
      2. 630 uL SYBR Fast
      3. 25.2 uL 10 uM AmpF
      4. 25.2 uL 10 uM AmpR-Ind20
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to plate layout
    4. File:PlateLayout-20170302-Padlock0601Test.png
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold

qPCR for post-Bglii digestion and gel images for all data continued tomorrow