Matt:LabNotes/2014-5-7
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ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA
- Continued from Matt:LabNotes/2014-4-30
- Test to see if rolonies quenched with TE (Tris + EDTA) are easier for padlock probes to hybridize to
- Quantify the amplification of Secondary Rolonization from Primary Rolonies
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
- Used Pinhole = 2.0
- Tried Pinhole = 1.0 for Pos9 the count of rolonies was 2,801 (80% of Pinhole 2.0)
Well | Position | Rolonies |
A3 | 1 | 930 |
A3 | 2 | 1562 |
A4 | 3 | 3134 |
A4 | 4 | 4352 |
A5 | 5 | 4997 |
A5 | 6 | 9189 |
D3 | 7 | 1272 |
D3 | 8 | 1062 |
D4 | 9 | 3480 |
D4 | 10 | 3758 |
D5 | 11 | 3315 |
D5 | 12 | 1909 |
File:MALAT1 2ndRol Results.JPG
Conclusion
- Padlock probe capture (followed by RCA) of primary rolonies greatly amplifies signal
- EDTA does not help with padlock probe capture
- Increasing concentration above 50pM does not improve 2nd rolony yield