Matt:LabNotes/2014-9-8: Difference between revisions
Jump to navigation
Jump to search
>Mzcai mNo edit summary |
>Mzcai mNo edit summary |
||
Line 36: | Line 36: | ||
*Add 200ul 1M Tris pH 8.0 at RT for 30min | *Add 200ul 1M Tris pH 8.0 at RT for 30min | ||
*Wash with 1X PBS twice and store in PBS | *Wash with 1X PBS twice and store in PBS | ||
===Imaging=== | |||
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 |
Revision as of 01:43, 12 September 2014
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
Imaging
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