Daniel:Notebook/ComboLock/2016-10-5: Difference between revisions

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Revision as of 19:49, 5 October 2016

==

Back to Calendar

Protocol

  C Probes-VIM (C1+C2) C Probes-SOD1 (C1+C2) Template
Sample 1 X X X
Sample 2 X X  
Sample 3     X
  1. Template-Bead Binding
  2. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
  3. Apply magnet for 30 sec and remove supernatant
  4. Add 2 uL template oligo per sample to bead solution; incubate at RT for 5 min
  5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  • C Probe Hybridization
    1. Combine 1 uL of each C probe and 3 uL CPH 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
  • Latch and Padlock Hybridization
    1. Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube
    2. Heat at 65C for 5 minutes
    3. Chill on ice for 3 minutes
    4. Add sample to prepared strep beads; vortex to suspend
    5. Incubate for 30 min at 37C with intermittent mixing
    6. Apply magnet and remove supernatant
    7. Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
    8. Repeat wash step above
    9. Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
    10. Resuspend with 20 uL 1x Amp Ligase buffer
  • Circularization
    1. Prepare 30 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight; continued tomorrow