Daniel:Notebook/ComboLock/2016-11-1

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Revision as of 17:59, 1 November 2016 by >Djacobse (→‎Protocol)
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Circularization Optimization Testing

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Sample Matrix

Sample Polymerase Incubation Time RCA Y/N
Sample 1 (AB) Klentaq ON Y
Sample 2 (AB) Klentaq ON N
Sample 3 (AB) Klentaq 2 hr Y
Sample 4 (AB) Klentaq 2 hr N
Sample 5 (AB) Phusion ON Y
Sample 6 (AB) Phusion ON N
Sample 7 (AB) Phusion 2 hr Y
Sample 8 (AB) Phusion 2 hr N

Protocol

  1. Circularization (all samples)
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 30 uL exonuclease I (20 units/μL) and 6 uL exonuclease III (100 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. Rolling Circle Amplification (Samples 1,3,5,7)
    1. Prepare samples in new tube according to following table
    2. Reagent uL Added Master Mix (8.1X)
      Template 5 NA
      RCA Primer (100 uM) 2.5 20.25
      dNTP (1 mM) 0.8 6.48
      10X Buffer 2 16.2
      Phi29 1 8.1
      nfH2O 8.7 70.47
      Total 20 121.5
    3. qPCR
      1. Set up reaction according to table below
      2. Make master mixes according to following recipes
        1. 54.25 uL nfH2O
        2. 77.5 uL SYBR Fast
        3. 1.55 uL AmpF
      3. Add 43 uL master mix to each well
      4. 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
    4. TBE Gel
      1. Mix 48 uL TBE, 12 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