Daniel:Notebook/ComboLock/2017-2-9
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Beads Binding Test
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Since this should only take a day (now that I have the proper materials), I want to test something: the beads binding. I want to see at what temp the beads lose their binding ability and at what temp we lose C probes for that matter. To that extent, I have planned the following experiment. It will cover incubation temperatures ranging from RT to 90C and will include a No C probe and no Template negative controls. I will also use positive controls in the qPCR (straight C probes and straight template).
Sample Matrix and Workflow
Sample Matrix
Workflow Diagram
File:20170208-BeadsTest-Workflow.png
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
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 |