Daniel:Notebook/ComboLock/2017-3-25

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Revision as of 23:35, 30 March 2017 by >Djacobse (→‎RCA Protocol)
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RCA Test[edit]

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This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the T4 experiments and the circular product production. I will be using that circular product to analyze the effectiveness of RCA.

Linear Product Production (Continued from yesterday)[edit]

  1. TBE gel
    1. Mix 64 uL TBE and 16 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample/ladder
    3. Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
    4. File:GelLanes-20170325-LinearProduct.png
    5. Add 10 uL mix to gel lanes
    6. Run gel for 24 minutes at 230V
    7. Stain with 2 uL SYBR gold for 3 minutes
    8. Rinse and image in gel doc

File:2017-03-25-LinearProductProduction.png

Still too much circular product present. I'll try one more time to make it linear.

Linear Product Production[edit]

This time we'll start with the circular product and chop it.

  1. Bglii digest
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol
      Circular Product (1X) 4
      Bglii Cutter Primer (100 uM) 2
      10X Buffer 3.1 2
      Bglii 1
      nfH2O 11
      Total 20
    3. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 4 hr 37C; When this cycle starts add 1 uL Bglii
      5. Heat kill with 65C for 20 min

    Continued tomorrow