Revision as of 02:00, 19 May 2014 by >Mzcai
MALAT1 Rolonies
Procedure
- 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
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
- Wash with 1X PBS twice
- Quench with 1M Tris pH 8.0 @RT for 30min
- Wash with 1X PBS twice
Detection
- 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
- Wash with H2O twice
Results
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
- Although conditions were exactly the same, 10 times more rolonies in C2 well
- Guessing this is because cells in C2 were denser (more confluent)
Example BF Image of B4 vs C2