Revision as of 18:48, 27 May 2014 by >Mzcai
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
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[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
- 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
Cell density very important in number of artificial rolonies that can be made