Matt:LabNotes/2014-4-9
Jump to navigation
Jump to search
Efficiency of Ampligase in situ Test
- Samples are wells F3-F10 from Matt:LabNotes/2014-3-31
- 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 it's possible 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 1uM ppMALAT1
- Add 50ul 1uM ppMALAT1 for 20hr @45C in 1X Ampligase buffer +/- 0.5U/ul Ampligase
F3-F6: -Ampligase
Component | Volume |
10X Ampligase Buffer | 20 |
100uM ppMALAT1 | 2 |
H2O | 178 |
Total | 200 |
F7-F10: + Ampligase
Component | Volume |
10X Ampligase Buffer | 20 |
100uM ppMALAT1 | 2 |
5 U/ul Ampligase | 20 |
H2O | 158 |
Total | 200 |
Digestion
- F3: Exo III
- F4: Exo I/III
- F5:
- F6:
- F7:
- F8: Exo I/III
- F9: Exo III
- F10: