Daniel:Notebook/ComboLock/2017-1-6

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Revision as of 01:19, 6 January 2017 by >Djacobse (→‎Protocol)
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Version 2 Oligos

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The version 2 oligos have several changes. The main is that they are shorter and use a UMI on the padlock instead of the latch. I will test these oligos and the new protocol. I'm also going to check

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-Phase 1

  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 following into a 0.2 mL tube
    2.   uL per Sample uL Total
      PCCA 1 8
      Mirror C Probe B reaction 2 16
      Wash buffer 2 16
      Total 5 40
    3. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    4. Add 5 uL probe mixture to beads
    5. Incubate at 40C for 1 hour with agitation; start part 3 after incubation begins
    6. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    7. Repeat wash step above
    8. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant

    Protocol-Phase 2

  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. Incubate at 37C for 30 minutes
  7. Circularization
    1. Prepare 8.5X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (8.5x) (uL)
      NAD+ 5 mM 40 nmol 8 68
      dNTP 1 mM 600 pmol 0.6 5.1
      Betaine 5 M 15 umol 3 25.5
      10X AmpLigase Buffer 10X 1X 2 17
      Amp Ligase 5 U/uL 10 U 2 17
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 27.2
      nf H2O     1.2 8.4
      Total     20 140

      Buffers

      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