Daniel:Notebook/ComboLock/2016-10-5: Difference between revisions
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C Probe Hybridization
Latch and Padlock Hybridization
Circularization
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Revision as of 19:49, 5 October 2016
==
Protocol
C Probes-VIM (C1+C2) | C Probes-SOD1 (C1+C2) | Template | |
Sample 1 | X | X | X |
Sample 2 | X | X | |
Sample 3 | X |
- Template-Bead Binding
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL template oligo per sample to bead solution; incubate at RT for 5 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Combine 1 uL of each C probe and 3 uL CPH buffer into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- 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
- Heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C with intermittent mixing
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Prepare 30 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight; continued tomorrow