Matt:LabNotes/2014-4-9: Difference between revisions
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>Mzcai m (→Detect) |
>Mzcai m (→Results) |
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===Results=== | ===Results=== | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Position''' | |||
| align="center" style="background:#f0f0f0;"|'''Well''' | |||
| align="center" style="background:#f0f0f0;"|'''Ampligase''' | |||
| align="center" style="background:#f0f0f0;"|'''Digestion''' | |||
| align="center" style="background:#f0f0f0;"|'''Rolonies''' | |||
|- | |||
| 5||F3||-||Exo III||203 | |||
|- | |||
| 6||F4||-||Exo I/III||146 | |||
|- | |||
| 7||F5||-||-||302 | |||
|- | |||
| 8||F6||-||-||230 | |||
|- | |||
| 9||F7||+||-||71 | |||
|- | |||
| 10||F8||+||Exo I/III||0 | |||
|- | |||
| 11||F9||+||Exo III||174 | |||
|- | |||
| 12||F10||+||-||195 | |||
|} | |||
===Conclusion=== | ===Conclusion=== |
Revision as of 18:21, 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
Results
Position | Well | Ampligase | Digestion | Rolonies |
5 | F3 | - | Exo III | 203 |
6 | F4 | - | Exo I/III | 146 |
7 | F5 | - | - | 302 |
8 | F6 | - | - | 230 |
9 | F7 | + | - | 71 |
10 | F8 | + | Exo I/III | 0 |
11 | F9 | + | Exo III | 174 |
12 | F10 | + | - | 195 |