Matt:LabNotes/2014-4-15
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Efficiency of Ampligase in situ Test: Second Try
- Previously results were not good enough to draw clear conclusion
- Samples are wells A5, D2, D3, D4, D5 from Matt:LabNotes/2014-4-5
- General idea: Will use Exo I & Exo III OR Exo III only to digest non-ligased padlock probes, then use dcProbe2RevComp to hybridize to padlock probe and detect
- Exo III only because Exo I will degrade all rolonies, leaving the padlock probe (even after ligation) nothing to attach to so it'll be washed away and not detected
- Ligation with Ampligase should protect the padlock probes from digestion, hence comparing wells +Exo with those -Exo will show what percentage of padlock probes are ligated
Hybridize 10nM ppMALAT1
- Add 100ul 10nM ppMALAT1 for 24hr @45C in 1X Ampligase buffer +/- 0.5U/ul Ampligase
A5&D2: -Ampligase
Component | Volume |
10X Ampligase Buffer | 20 |
1uM ppMALAT1 | 2 |
H2O | 178 |
Total | 200 |
D3,D4,&D5: + Ampligase
Component | Volume |
10X Ampligase Buffer | 30 |
1uM ppMALAT1 | 3 |
5 U/ul Ampligase | 30 |
H2O | 237 |
Total | 300 |
Digestion
- Wash with 2X SSC once
- Add 100ul Exonuclease mix to each well
- Incubate @37C for 3 hrs
Exo I/III: D3
Component | Volume |
10X Exo III Buffer | 10 |
Exo I | 5 |
Exo III | 5 |
H2O | 80 |
Total | 100 |
Exo III: D2&D4
Component | Volume |
10X Exo III Buffer | 20 |
Exo III | 10 |
H2O | 170 |
Total | 200 |
Control: A5&D5
Component | Volume |
10X Exo III Buffer | 20 |
H2O | 180 |
Total | 200 |
Detect
- Wash with 2X SSC twice
- Add 100ul 1uM dcProbe2RevComp-Cy3 (ATTO 488 dye)
- Pre-heated to 75C
- Wash with 2X SSC twice