Matt:LabNotes/2014-4-9: Difference between revisions
Jump to navigation
Jump to search
>Mzcai |
>Mzcai m (→Digestion) |
||
Line 49: | Line 49: | ||
*F9: Exo III | *F9: Exo III | ||
*F10: | *F10: | ||
Exo I/III: F4 & F8 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10X Exo III Buffer||10 | |||
|- | |||
| Exo I||5 | |||
|- | |||
| Exo III||5 | |||
|- | |||
| H2O||80 | |||
|- | |||
| Total||100 | |||
|} | |||
Exo III: F3 & F9 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10X Exo III Buffer||10 | |||
|- | |||
| Exo III||5 | |||
|- | |||
| H2O||85 | |||
|- | |||
| Total||100 | |||
|} | |||
Control: F5, F6, F7 & F10 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10X Exo III Buffer||20 | |||
|- | |||
| H2O||180 | |||
|- | |||
| Total||200 | |||
|} |
Revision as of 23:08, 10 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 20hr @45C (7:00pm- ) 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
- F3: Exo III
- F4: Exo I/III
- F5:
- F6:
- F7:
- F8: Exo I/III
- F9: Exo III
- F10:
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 |