Daniel:Notebook/ComboLock/2016-11-15
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BSA Beads Test
Given the results from the primers test and most recent cells test, it seems that using the primer from Chris with RCA has provided a more consistent positive control. I'm going to repeat the experiment from November 3rd regarding the BSA-strep beads.
Protocol
Sample | BSA Ab | BSA-Biotin | Dilution | Dilution Amount |
Sample 1 (AB) | X | X | 0 | 2 ug |
Sample 2 (AB) | X | NA | None | |
Sample 3 (AB) | X | 0 | 2 ug | |
Sample 4 (AB) | X | X | 2 | 20 ng |
Sample 5 (AB) | X | X | 4 | 200 pg |
- 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 2 mg/mL biotin-BSA 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 2 uL of BSA Ab-AB mix (75 ug/uL stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Incubate at RT for 1.5 hours with agitation
- Add 100 uL of wash buffer
- 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
- Latch and Padlock Hybridization
- Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0201 to each sample to a fresh 0.2 mL tube
- Tech Rep A-Latch0001
- Tech Rep B-Latch0002
- Heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C with intermittent mixing
- Add 100 uL wash buffer; take 10 uL as a time point
- Apply magnet and remove supernatant; Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend
- Apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 55C for 2 hours
- Heat kill by incubating at 94C for 5 minutes
- Rolling Circle Amplification (Samples 1,3,5,7; Hold samples 2,4,6,8 at 4C)
- Prepare 8.1X Master mix according to table below
- Add sample to new reaction tubes according to following table
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes
- Hold at 10C; continued tomorrow
Reagent | uL Added | Master Mix (10.2X) |
Template | 5 | NA |
RCA Primer (100 uM) | 2.5 | 25.5 |
dNTP (1 mM) | 0.8 | 8.16 |
10X Buffer | 2 | 20.4 |
Phi29 | 1 | 10.2 |
nfH2O | 8.7 | 88.74 |
Total | 20 | 153 |
Library Prep
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