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

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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)

  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

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