Matt:LabNotes/2014-9-8
Jump to navigation
Jump to search
in situ Padlock Probe Capture Temp: 45C vs 60C[edit]
Procedure[edit]
- 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
Imaging[edit]
Dish #1 (45C)
20X Objective Laser 4% Gain 680 Offset -1.05% Pinhole 1.00 Zoom 1.00 4096x4096 600Hz
Pos1: (67.8,41.12) z: 1552.95-1572.66 Pos2: (67.31,42.03)z: 1559.43-1578.20 Pos3: (65.91,40.54)z: 1565.21-1585.86
Dish #2 (60C)
Pos1: (71.5,40.12) z: 1361.76-1380.53 Pos2: (71.02,45.03) z: 1313.81-1334.46
Result[edit]
- Using PISA to count features
' | 45C | 45C | 60C | 60C |
1st Rolonies | COL1A1 Rolonies | 1st Rolonies | COL1A1 Rolonies | |
Position 1 | 3,268 | 1,887 (58%) | 1,751 | 1,973 (113%) |
Position 2 | 4,341 | 2,259 (52%) | 2,342 | 1,820 (78%) |
Position 3 | 3,891 | 1,609 (41%) |
Example images[edit]
File:20140911 45C Pos2 1stRolonies.PNG File:20140911 45C Pos2 COL1A1Rolonies.PNG
Conclusion[edit]
- 60C may be more effective for Ampligase step but hard to compare because there were a different number of starting primary rolonies
- Since 60C is definitely not worse we should continue with 60C padlock probe capture until a problem occurs
- ppCOL1A1 is highly localized to nucleus, even though primary rolonies are evenly distributed in cell