Matt:LabNotes/2014-4-9: Difference between revisions
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>Mzcai m (→Digestion) |
>Mzcai m (→Detect) |
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Line 93: | Line 93: | ||
**Pre-heated to 75C then snap-cooled before adding | **Pre-heated to 75C then snap-cooled before adding | ||
*Wash with 2X SSC twice | *Wash with 2X SSC twice | ||
*Images saved in '''4- | *Images saved in '''4-10-2014''' | ||
===Results=== | ===Results=== | ||
===Conclusion=== | ===Conclusion=== |
Revision as of 02:50, 11 April 2014
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 2uM ppMALAT1
- Add 50ul 2uM ppMALAT1 for 21.5hr @45C (7:00pm-4:30pm ) in 1X Ampligase buffer +/- 0.5U/ul Ampligase
F3-F6: -Ampligase
Component | Volume |
10X Ampligase Buffer | 20 |
200uM ppMALAT1 | 2 |
H2O | 178 |
Total | 200 |
F7-F10: + Ampligase
Component | Volume |
10X Ampligase Buffer | 20 |
200uM ppMALAT1 | 2 |
5 U/ul Ampligase | 20 |
H2O | 158 |
Total | 200 |
Digestion
- Wash with 2X SSC once
- Add 50ul Exonuclease mix
- Incubate @37C for 2 hrs
Exo I/III: F4 & F8
Component | Volume |
10X Exo III Buffer | 10 |
Exo I | 5 |
Exo III | 5 |
H2O | 80 |
Total | 100 |
Exo III: F3 & F9
Component | Volume |
10X Exo III Buffer | 10 |
Exo III | 5 |
H2O | 85 |
Total | 100 |
Control: F5, F6, F7 & F10
Component | Volume |
10X Exo III Buffer | 20 |
H2O | 180 |
Total | 200 |
Detect
- Wash with 2X SSC twice
- Add 50ul 1uM dcProbe2RevComp-Cy3 (ATTO 488 dye)
- Pre-heated to 75C then snap-cooled before adding
- Wash with 2X SSC twice
- Images saved in 4-10-2014