Matt:LabNotes/2014-9-6
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"Tertiary" Rolony Synthesis
- Hypothesis: We can increase the number of gene-specific rolonies (previously known as secondary rolonies) by repeating padlock probe capture and rolonization on samples with primary and secondary rolonies
- There should be more targets for the padlock probes to capture since all primary rolonies are still fixed and 100% of secondary rolonies will be possible targets for padlock probes
- May increase amplification bias
Procedure
- PGP1F sample from 4/14/14
- Add 100ul Padlock Probe Mix (14pM per padlock probe)
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
908 nM Agi26k0gap | 18.6 |
H2O | 61.4 |
Total | 100 |
- Incubate at 37C for 30min
- Incubate at 45C for 26hrs
- Wash with 1X PBS twice
- Add 200ul 0.5 uM FISSEQ_ppRCA primer in 2X SSC (pre-heated to 75C) and incubate at 45C for 30min
- Wash with 2X SSC twice
- Add 200ul RCA Mix and incubate at 30C for 16hrs
- H2O : 172uL
- 10x Buffer : 20uL
- dNTP : 2uL
- a-dUTP : 4uL
- Phi29 (100 U/µl) : 2uL
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
- Wash with 1X PBS twice
- Add 200ul 1M Tris pH 8.0 at RT for 30min
- Wash with 1X PBS twice and store in PBS
7 Cycle 3-Dye Decoding
- Saved in 9-9-2014