Daniel:Notebook/ComboLock/2017-2-9
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Beads Binding Test[edit]
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[edit]
Sample Matrix
Sample | 1 (AB) | 2 (AB) | 3 (AB) | 4 (AB) | 5 (AB) | 6 (AB) | 7 (AB) | 8 (AB) | 9 (AB) | 10 (AB) |
Condition | RT | No Template | 30C | 40C | 50C | 60C | 70C | 80C | 90C | No C Probe |
Workflow Diagram
File:20170208-BeadsTest-Workflow.png
Protocol[edit]
- 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; For "No Template" add 2 uL 2 mg/mL BSA-biotin
- 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 Testing
- Repeat following washes 5 times
- Add 100 uL wash buffer
- Set on magnet for 1 min; take 15 uL aside for testing
- Vortex and incubate for 5 minutes at appropriate incubation temperature
- Set on magnet for 1 min; remove supernatant completely
Reagent | uL per Sample | uL Total (Samples+NT) | uL Total (No C Probe) |
PCCA-03 (10 uM) | 1 | 18 | 0 |
PCCB-03 (10 uM) | 1 | 18 | 0 |
Wash buffer | 3 | 54 | 10 |
Total | 5 | 90 | 10 |
Ran out of time; continued tomorrow