Daniel:Notebook/ComboLock/2017-3-13
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Bolt Test (Started Wednesday March 8)
Protocol
Sample Matrix
Sample | Condition |
Sample 5 (AB) | No Ligase |
Sample 6 (AB) | Normal |
- 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 10 uM 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
- C Probe Hybridization
- Combine following into a 0.2 mL tube
- 4 uL each PCCA-03 and PCCB-03 (10 uM each)
- 12 uL wash buffer
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Repeat wash step above
Protocol-Part 2-LB hybridization & ligation
- Bolt and Latch Hybridization/Ligation
- Set up the following master mix
- Add 19 uL probe mix to each sample
- Incubate at 55C for 10 minutes
- Incubate at 25C for 10 minutes
- Lower the temperature to 20C; wait 30 seconds then take off incubator
- Add 1 uL T4 Ligase to each reaction
- Incubate at RT for 15 min
- Heat kill enzyme by incubating at 65C for 10 minutes
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (4.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 16.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 4.2 |
Lock Oligo | 10 uM | 10 pmol | 1 | 4.2 |
nfH2O | NA | NA | 13 | 54.6 |
Total | 19 | 79.8 |