Daniel:Notebook/ComboLock/2017-4-12

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RCA Double Deuce (Started Yesterday)[edit]

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Protocol[edit]

  1. Bglii digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (13.2X)
      RCA Rxn 7 0
      10X Buffer 3.1 2 26.4
      Bglii 1 0
      nfH2O 10 132
      Total 20 158.4
    3. Aliquot 12 uL master mix into new tube
    4. Add 7 uL appropriate sample
    5. Mix with vortexer, spin down, and add 1 uL Bglii
    6. Incubate 2 hr 37C
    7. Heat kill with 65C for 20 min
  2. TBE Gel
    1. Mix 120 uL 10X TBE and 40 uL 6X dye
    2. Aliquot 8 uL TBE/dye mix onto parafilm per sample
    3. Add 4 uL sample to sample lanes
    4. Mix 8 uL 1X TBE, 2 uL 6X dye, and 1.5 uL 25bp ladder for ladder sample
    5. Add 10 uL from aliquots into appropriate gel lanes
    6. Run gel for 25 minutes at 250V
    7. Stain with 2 uL SYBR gold for 3 min
    8. Rinse and image in gel doc