Daniel:Notebook/ComboLock/2017-3-1
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Padlock0601 Test
Testing the new arrival of padlock0601, which also contains a restriction enzyme cutting site (Bam)
Protocol-Part 1
- 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
- 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
- Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
Reagent | uL per Sample | uL Total (Samples+NT) | uL Total (No Phosphate) | uL Total (No C Probe) |
PCCA-03 (10 uM) | 1 | 4 | 2 | 0 |
PCCB-03 (10 uM) | 1 | 4 | 2*** | 0 |
Wash buffer | 3 | 12 | 6 | 10 |
Total | 5 | 20 | 10 | 10 |