Daniel:Notebook/ComboLock/2017-6-14
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Antibody Detection Test-Positive Control[edit]
This will be a basic test of the antibody detection.
Protocol[edit]
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No Antibodies |
- Template-Bead Binding
- Suspend 2.5 uL (25 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Prepare following mixes
- 4X mix (samples 1,3): 80 uL Dynabuffer, 72 uL nfH2O, 8 uL BSA-biotin 0.2 mg/mL (6 pmol, 6X excess)
- 2X mix (sample 2): 40 uL Dynabuffer, 36 uL nfH2O, 4 uL BSA-biotin 0.2 mg/mL (6 pmol, 6X excess)
- Add 40 uL per sample and incubate at RT for 15 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Antibody hybridization
- Combine following into 0.2 mL tubes
- Add probes to cell sample for final concentration of 0.5 nM
- Incubate 1.5 hours at RT
- Wash beads with 100 uL wash buffer with 0.05% Tween 20; apply magnet and remove supernatant
- Repeat wash step above
- Repeat wash step above
******
Reagent | uL per Sample | Exp uL Total (4X 1,2) | No Ab uL Total (2X-Sample 3) |
Ab-A (75 ug/mL-0.5 uM) | 2 | 8 | 0 |
Ab-B (75 ug/mL-0.5 uM) | 2 | 8 | 0 |
PBS+EDTA (2X+10 mM) | 10 | 40 | 20 |
nfH2O | 6 | 24 | 20 |
Total | 20 | 80 | 20 |
Protocol-Part 2[edit]
- Latch and Bolt 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 16C overnight Continued tomorrow
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (6.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 24.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 6.2 |
Bolt Oligo | 10 uM | 10 pmol | 1 | 6.2 |
Dephosphorylated Bolt | 2.5 uM | 10 pmol | 8 | 0 |
nfH2O | NA | NA | 13 | 80.6 |
Total | 19 | 117.8 |