Daniel:Notebook/ComboLock/2016-12-17

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Version 2 Oligos[edit]

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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. I won't have time today to actually run this, so the following protocol will only be for reference to start when I get back.

Since I didn't have time before break this is just the suggested protocol to use once I get back.

Sample Matrix[edit]

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

Protocol[edit]

  1. Phosphorylation-MirrorC Probe B (PCCB-02)
    1. Set up reaction according to table
    2. Reagent Stock Conc Final Conc./Amount uL added
      T4 Polynucleotide Kinase Buffer 10X 1X 2
      ATP 10 mM 1 mM 2
      PCCB-02 10 uM 100 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-Lock oligo
    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
      Lock0201 10 uM 10 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
  5. Lock and Latch Hybridization/Ligation
    1. Set up the following master mix
    2. Reagent Stock Conc Final Conc./Amt uL added Master Mix (8.2X)
      T4 Ligase Reaction Buffer 10X 1X 2 16.4
      Latch Oligo 10 uM 10 pmol 1 8.2
      Lock Oligo Phosphate Reaction (5 uM Lock) 10 pmol 2 16.4
      nfH2O NA NA 14 114.8
      Total     19 155.8
    3. Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201
    4. Incubate at 95C for 5 min
    5. Lower the temperature to 20C; wait 30 seconds then take off incubator
    6. Add 1 uL T4 Ligase to each reaction
    7. Incubate at RT for 30 min
    8. Heat kill enzyme by incubating at 65C for 10 minutes
    9. Wash twice with 100 uL wash buffer
  6. Padlock Hybridization
    1. Make following master mix
      1. 16.4 uL 10 uM padlock0401
      2. 16.4 uL Amp Ligase 10X reaction buffer
      3. 131.2 uL nfH2O
    2. Resuspend sample in 20 uL padlock buffer
    3. Circularization
      1. Prepare 9X uL Phusion mix
      2. Buffers[edit]

        Wash Buffer

        Reagent Stock Final Dilution Amt in 20 mL
        NaCl 1.5 M 0.5 M 3 6.66 mL
        Tris-HCl 500 mM 20 mM 25 800 uL
        EDTA 0.5M 1 mM 500 40 uL
        nf H2O NA NA NA 12.5 mL