Daniel:Notebook/ComboLock/2016-11-28

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Circularization Test 2[edit]

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This is the repetition of RCA step from Nov 23, but with my new RCA primer. It should target the LinkerL region of the circular product.

Protocol[edit]

  1. Rolling Circle Amplification
    1. Prepare 7X Master mix according to table below
    2. Add 15 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (7X)
      Template 5 NA
      RCA Primer (100 uM) 2.5 17.5
      dNTP (1 mM) 0.8 5.6
      10X Buffer 2 14
      Phi29 1 7
      nfH2O 8.7 60.9
      Total 20 105
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step
  2. qPCR
    1. Make master mix according to following recipe
      1. 149.1 uL nfH2O
      2. 177.5 uL SYBR Fast
      3. 7.1 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section
    4. Sample Condition Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 1A Sample A1 20 2 25 1 1 21 50
      Sample 1B Sample A2 20 2 25 1 1 21 50
      Sample 2A No Template A3 21 2 25 1 1 21 50
      Sample 2B No Template A4 21 2 25 1 1 21 50
      Sample 3A No C Probe A5 22 2 25 1 1 21 50
      Sample 3B No C Probe A6 22 2 25 1 1 21 50
      NTC qPCR Primers Only A7 23 2 25 1 1 23 50
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold
  3. TBE Gel
    1. Mix 72 uL TBE, 18 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]