Daniel:Notebook/ComboLock/2017-1-20

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Revision as of 21:49, 20 January 2017 by >Djacobse (→‎Random Hexamer RCA)
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Version 3 Oligos (Started Thursday, Jan 13)

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RCA Test-Adpaters

From yesterday's results I'm going to try the short RCA again, this time using the adapter regions as primers. I'll test using Lock0301 and Latch0301, since those should only amplify if circular product exists.

  1. Rolling Circle Amplification
    1. Prepare 8.5X Master mix according to table below
    2. Add 12.5 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (8.5X)
      Template 5 NA
      RCA Primer (Latch0301 or Lock0301); 10 uM) 2.5 21.25
      dNTP (1 mM) 0.8 6.8
      10X Phi29 Buffer 2 17
      Phi29 1 8.5
      nfH2O 8.7 73.95
      Total 20 127.5
    5. Incubate at 37C for 30 minutes
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step
  2. Qubit Measure
    1. Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)
    2. Add 190 uL dye/buffer and 10 uL standard to standard tubes
    3. Add 199 uL dye/buffer and 1 uL sample to sample tubes
    4. Measure in Qubit

Results

Random Hexamer RCA

  1. Rolling Circle Amplification
    1. Prepare 8.5X Master mix according to table below
    2. Add 12.5 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (8.5X)
      Template 5 NA
      Random Hexamer (100 uM) 2.5 11.25
      dNTP (1 mM) 0.8 3.6
      10X Phi29 Buffer 2 9
      Phi29 1 4.5
      nfH2O 8.7 39.15
      Total 20 67.5