Daniel:Notebook/ComboLock/2017-4-4

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Revision as of 17:22, 4 April 2017 by >Djacobse (→‎Bgl Digestion-Excessive)
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RCA Test (Started March 24)

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Bgl Digestion-Excessive

  1. Bgl Digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (6.2X)
      RCA Rxn 8 0
      10X Buffer 3.1 2 12.4
      Bglii 1 0
      nfH2O 9 55.8
      Total 20 68.2
    3. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 1 hr 37C; When this cycle starts add 2 uL Bglii
      5. Heat kill with 65C for 20 min
  2. TBE Gel
    1. Mix 60 uL 10X TBE and 20 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot
    3. Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot
    4. Add 10 uL mix to gel lanes
    5. Run gel for 24 minutes at 230V
    6. Stain with 2 uL SYBR gold for 3 minutes
    7. Rinse and image in gel doc