Daniel:Notebook/ComboLock/2016-12-16

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Revision as of 18:05, 16 December 2016 by >Djacobse (→‎Protocol)
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Overnight Capture (Started Yesterday)

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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. 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
    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
      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

      Version 2 Oligos

      The version 2 oligos have several changes. The first is that they are shorter. The C probes have a 20bp target matching region (from 24bp) and the adapters are 15bp (from 18bp). This makes the C probes exactly 60bp which means they are regular oligos (not Ultramers) and can be received one day after ordering. A second improvement is to include the idea of the lock oligo. Note, this is not a Lock nucleic acid, it's just a name. The lock oligo fits in between the two C probes, spanning the gap between them. In conjunction with the latch oligo, this theoretically means that we can ligate the C probes together without using polymerase. This will allow the padlock probe to act as a standard gap-fill padlock (instead of 2 small gaps) since it is one medium sized gap.

      Sample Matrix

      Sample Condition
      Sample 1 (AB) Normal
      Sample 2 (AB) No Template
      Sample 3 (AB) No C probes
      Sample 4 (AB) No phosphate activation

      Protocol

      1. Phosphorylation-MirrorC Probe B (PCCB-02)
        1. Set up reaction according to table
        2. Reagent Stock Conc Final Conc./Amount uL added
          T4 DNA Ligase Buffer 10X 1X 2
          ATP 10 mM 1 mM 2
                 
          PCCB-02 10 uM 150 pmol total 10
          T4 DNA Kinase 10 U/uL 10 U 1
          nfH2O NA NA 5
          Total     20
        3. Incubate at 37C for 30 min
        4. Heat kill enzyme with 15 minutes at 65C
      2. Template-Bead Binding
        1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
        2. Apply magnet for 30 sec and remove supernatant
        3. Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
        4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
      3. C Probe Hybridization
        1. Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
        2. Heat C probes to 90C for 5 min; Chill probes on ice to quench
        3. Add 5 uL probe mixture to beads
        4. Incubate at 40C for 1 hour with agitation; start part 3 after incubation begins
        5. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
        6. Repeat wash step above
        7. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
      4. Phosphorylation
        1. Set up reaction according to table