Daniel:Notebook/ComboLock/2017-3-7

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Padlock0601 Test (Started Wed March 1)[edit]

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Protocol-Part 2 (Protocol Started Yesterday)[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) 0 0
      10X Buffer 3.1 2 16.4
      Bglii 1 8.2
      nfH2O 12 98.4
      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 16.2X qPCR master mix according to following recipe
      1. 340.2 uL nfH2O
      2. 405 uL SYBR Fast
      3. 16.2 uL 10 uM AmpF
      4. 16.2 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-20170307-Padlock0601-MaxRCA.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
  3. TBE Gel
    1. Mix 160 uL TBE, 40 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 23 minutes at 230V
    6. Open gel and stain with 2 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Protocol-Part 3-Post Bglii[edit]

  1. qPCR
    1. Make 9.2X qPCR master mix according to following recipe
      1. 193.2 uL nfH2O
      2. 230 uL SYBR Fast
      3. 9.2 uL 10 uM AmpF
      4. 9.2 uL 10 uM AmpR-Ind21
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to plate layout
    4. File:PlateLayout-20170307-MaxRCA-BgliiDigest.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
  2. TBE Gel
    1. Mix 96 uL TBE, 24 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 23 minutes at 230V
    6. Open gel and stain with 2 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc


Results[edit]