Matt:LabNotes/2014-5-7: Difference between revisions
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>Mzcai (Created page with "==ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA== ===Procedure=== *Add 100ul ppMALAT1_dcProbe1 with 0.5 U/ul Ampligase **A3&D3: 10pM **A4&D4: 50pM **A5&D5: 2uM **Incu...") |
>Mzcai mNo edit summary |
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*Wash 2X SSC twice | *Wash 2X SSC twice | ||
*Image | *Image | ||
**Used Pinhole: 2.0 | |||
===Results=== | ===Results=== | ||
*Saved in 5-8-2014 | *Saved in 5-8-2014 |
Revision as of 22:53, 8 May 2014
ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA
Procedure
- Add 100ul ppMALAT1_dcProbe1 with 0.5 U/ul Ampligase
- A3&D3: 10pM
- A4&D4: 50pM
- A5&D5: 2uM
- Incubate @45C for 24 hours
10pM
Component | Volume |
10X Ampligase Buffer | 20 |
5 U/ul Ampligase | 20 |
1nM ppMALAT1_dcProbe1 | 2 |
H2O | 158 |
Total | 200 |
50pM
Component | Volume |
10X Ampligase Buffer | 20 |
5 U/ul Ampligase | 20 |
1nM ppMALAT1_dcProbe1 | 10 |
H2O | 150 |
Total | 200 |
2uM
Component | Volume |
10X Ampligase Buffer | 20 |
5 U/ul Ampligase | 20 |
100uM ppMALAT1_dcProbe1 | 4 |
H2O | 156 |
Total | 200 |
- Aspirate NO WASH
- Add RCA primer: 3ul FISSEQ_ppRCA in 597 2X SSC/30%
- Pre-heat to 60C
- Add 100ul @45C for 15min
- Wash 2X SSC twice
- Wash 0.1X SSC twice
- Add RCA mix
- Incubate 22hrs @ 30C
Component | Volume |
H2O | 513 |
10X Phi29 Buffer | 60 |
25mM dNTP | 6 |
2mM aa-dUTP | 12 |
Phi29 | 9 |
Total | 600 |
- Wash 1X PBS once
- 12ul BS(PEG)9 in 588ul 1X PBS
- Incubate @RT 1hr
- Wash 1X PBS twice
- Quench with 1M Tris pH 8.0 @RT 1hr
- Wash 1X PBS twice
- Add 1uM dcProbe1-Cy3 in 30% formamide + 2X SSC
- Preheat to 75C for 5min
- Incubate @RT for 10min
- Wash 2X SSC twice
- Image
- Used Pinhole: 2.0
Results
- Saved in 5-8-2014