Matt:LabNotes/2014-5-13
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MALAT1 Rolonies[edit]
- Goal is to make more artificial MALAT1 Rolonies for padlock probe hybridization testing
- Test to see the effect of increasing padlock probe concentration (10pM -> 100pM)
Procedure[edit]
- After fixing with 10% formalin 15min 37C & then sitting in 70% EtOH, Wash twice with 1X PBS
- Add 140ul 100nM MALAT1_template to wells C2 & B4
- Pre-heat to 85C
- Incubate @RT for 15 min
- Aspirate & Wash once with 1X PBS
- Capture with 100pM ppMALAT1_dcProbe1
- Incubate @45C for 24 hours
Component | Volume |
10X Ampligase Buffer | 20 |
1nM ppMALAT1_dcProbe1 | 20 |
5 U/ul Ampligase | 20 |
H2O | 140 |
Total | 200 |
- Aspirate NO WASH
- Add RCA primer: 1ul FISSEQ_ppRCA in 199ul 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 | 171 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
Phi29 | 3 |
Total | 200 |
- Wash with 1X PBS once
- 4ul BS(PEG)9 in 196ul 1X PBS
- Incubate 1hr @ RT
- Wash with 1X PBS twice
- Quench with 1M Tris pH 8.0 @RT for 30min
- Wash with 1X PBS twice
Detection[edit]
- Add 0.5uM dcProbe1-Cy3 in 30% formamide + 2X SSC
- Preheat to 75C for 5min
- Incubate @RT for 10min
- Wash with 2X SSC twice
- Image with Confocal (Saved in 5-16-2014)
- Laser 3% & Gain 550
- Pinhole = 1.0
- Strip with 80% formamide for 10min @RT
- Preheated to 75C
- Wash with H2O twice
Results[edit]
Position | Rolonies |
1 | 472 |
2 | 566 |
3 | 334 |
4 | 3221 |
5 | 2816 |
6 | 2813 |
- Comparing 3% vs 5% Laser
- Position1 Laser 5%: 905 rolonies
- Position1 Laser 3%: 472 rolonies
Conclusion[edit]
- Compared to using 10pM ppMALAT1
- Expect to see a ~10X increase in MALAT1 rolonies (from 100 to 1,000)
- Definitely increased in number of rolonies but wasn't consistent
- Although conditions were exactly the same, 10 times more rolonies in C2 well than B4
- Guessing this is because cells in C2 were denser (more confluent)
Example BF Image of B4 vs C2
B4 | C2 |
Position 2 | Position 5 |
File:Pos2 B4 MALAT1Rol dcProbe1 L3 G550 Cy3 z01 ch01.jpg | File:Pos5 C2 MALAT1Rol dcProbe1 L3 G550 Cy3 z01 ch01.jpg |
Cell density very important in number of artificial rolonies that can be made