Daniel:Notebook/ComboLock/2016-12-16

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Overnight Capture (Started Yesterday

Back to Calendar

Protocol

  1. Circularization
    1. Prepare 7X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (7x) (uL)
      NAD+ 5 mM 40 nmol 8 56
      dNTP 1 mM 600 pmol 0.6 4.2
      Betaine 5 M 15 umol 3 21
      10X AmpLigase Buffer 10X 1X 2 14
      Amp Ligase 5 U/uL 10 U 2 14
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 22.4
      nf H2O     1.2 8.4
      Total     20 140
    3. Add 20 uL Phusion mix to 20 uL of reaction
    4. Incubate at 60C for 2 hours
    1. Exonuclease Digestion
      1. Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)
      2. Add 2 uL to each sample, mix by swirling pipette tip
      3. Incubate for 1 hour at 37C
      4. Heat kill by incubating for 5 min at 95C
    2. 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; use template from both supernatant only and beads
      4. Sample Condition Fraction 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 Supernatant A1 20 2 25 1 1 21 50
        Sample 1B Sample Supernatant A2 20 2 25 1 1 21 50
        Sample 2A No Template Supernatant A3 21 2 25 1 1 21 50
        Sample 2B No Template Supernatant A4 21 2 25 1 1 21 50
        Sample 3A No C Probe Supernatant A5 22 2 25 1 1 21 50
        Sample 3B No C Probe Supernatant A6 22 2 25 1 1 21 50
        Sample 1A Sample Beads A1 20 2 25 1 1 21 50
        Sample 1B Sample Beads A2 20 2 25 1 1 21 50
        Sample 2A No Template Beads A3 21 2 25 1 1 21 50
        Sample 2B No Template Beads A4 21 2 25 1 1 21 50
        Sample 3A No C Probe Beads A5 22 2 25 1 1 21 50
        Sample 3B No C Probe Beads A6 22 2 25 1 1 21 50
        NTC qPCR Primers Only   A7 23 2 25 1 1 23 50
      5. Incubate at 37C for 3 hours
      6. Incubate at 65C for 10 minutes;Hold at 10C until next step
    3. qPCR
      1. Make master mix according to following recipe
        1. 151.2 uL nfH2O
        2. 180 uL SYBR Fast
        3. 7.2 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