Daniel:Notebook/ComboLock/2017-1-13
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Version 3 Oligos
The main difference between version 2 oligos and version 3 oligos is only adapter length. The version 3 oligos use the original 18bp adapters instead of the 15bp ones. This helps the annealing temperatures be high, which is better for enzymes such as Amp Ligase and Phusion. The other difference is that the version 3 oligos have phosphate groups on the 5'
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probes |
Sample 4 (AB) | No phosphate activation |
Protocol
- 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 |
PCCA-03 | 1 | 6 |
PCCB-03 | 1 | 6 |
Wash buffer | 3 | 18 |
Total | 5 | 30 |