Matt:LabNotes/2014-9-8
Jump to navigation
Jump to search
in situ Padlock Probe Capture Temp: 45C vs 60C
Procedure
- PGP1F Samples with 1st Rolonies prepared by Hosuk
- Fixed 9/6/14 and 1st Rolonies on 9/8/14
- Add 100ul Padlock Probe Mix
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
100uM ppCOL1A1 | 2 |
H2O | 78 |
Total | 100 |
- Incubate at 37C for 30min
- Incubate at 45C or 60C for 26hrs
- Wash with 1X PBS twice
- Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (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