Matt:LabNotes/2014-4-30
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MALAT1 Rolonies and EDTA[edit]
- Test to see if rolonies quenched with TE (Tris + EDTA) are easier for padlock probes to hybridize to
- Use artificial MALAT1 rolonies
- Quantify the amplification of Secondary Rolonization from Primary Rolonies
Procedure[edit]
- Add 140ul 100nM MALAT1_template to each well
- Pre-heat to 85C
- Incubate @RT for 15 min
- Aspirate & Wash once with 1X PBS
- Capture with 10pM ppMALAT1
- Incubate @45C for 20 hours
Component | Volume |
10X Ampligase Buffer | 60 |
1nM ppMALAT1 | 6 |
5 U/ul Ampligase | 60 |
H2O | 474 |
Total | 600 |
- 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
- To A3-A5: 1M Tris pH 8.0
- To D3-D5: 0.9M Tris + 50mM EDTA ph 8.0 (45mL 1M Tris + 5mL 500mM EDTA)
- Wash 1X PBS twice
- Add 1uM dcProbe2-Cy3 in 30% formamide + 2X SSC
- Preheat to 75C for 5min
- Incubate @RT for 10min
- Wash 2X SSC twice
- Image
- Strip w/ 80% formamide in 2X SSC
- Pre-heat to 75C
- Incubate @RT for 15min
- Wash with ddH2O twice
Results[edit]
- PISA wasn't able to count many images due to not reaching minimum number of features
- Used bwlabel to count features after applying a threshold of 9,000 to make a bw image
Well | Position | Rolonies counted by PISA | Rolonies counted by bwlabel (threshold 9000) |
A3 | 1 | N/A | 55 |
A3 | 2 | 140 | 141 |
A4 | 3 | N/A | 69 |
A4 | 4 | N/A | 104 |
A5 | 5 | N/A | 66 |
A5 | 6 | N/A | 127 |
D3 | 7 | N/A | 41 |
D3 | 8 | N/A | 51 |
D4 | 9 | N/A | 48 |
D4 | 10 | 137 | 146 |
D5 | 11 | 139 | 140 |
D5 | 12 | 128 | 127 |
- Continued on Matt:LabNotes/2014-5-7