Daniel:Notebook/ComboLock/2017-3-13

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Bolt Test (Started Wednesday March 8)

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Protocol

Sample Matrix

Sample Condition
Sample 5 (AB) No Ligase
Sample 6 (AB) Normal
  1. 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
  2. C Probe Hybridization
    1. Combine following into a 0.2 mL tube
      1. 4 uL each PCCA-03 and PCCB-03 (10 uM each)
      2. 12 uL wash buffer
    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
    5. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    6. Repeat wash step above
    7. Repeat wash step above

Protocol-Part 2-LB hybridization & ligation

  1. Bolt 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 5X 1X 4 32.8
      Latch Oligo 10 uM 10 pmol 1 8.2
      Lock Oligo 10 uM 10 pmol 1 8.2
      nfH2O NA NA 13 106.6
      Total     19 155.8
    3. Add 19 uL probe mix to each sample
    4. Incubate at 55C for 10 minutes
    5. Incubate at 25C for 10 minutes
    6. Lower the temperature to 20C; wait 30 seconds then take off incubator
    7. Add 1 uL T4 Ligase to each reaction
    8. Incubate at RT for 15 min
    9. Heat kill enzyme by incubating at 65C for 10 minutes