Matt:LabNotes/2014-5-16: Difference between revisions
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>Mzcai (Created page with "==Detecting ppMALAT1 Hybridization== *Use sample from Matt:LabNotes/2014-5-7 **A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2-RevComp in one step **D3 & D4: Hybridi...") |
>Mzcai mNo edit summary |
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*Use sample from [[Matt:LabNotes/2014-5-7]] | *Use sample from [[Matt:LabNotes/2014-5-7]] | ||
**A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM | **A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2RevComp in one step | ||
**D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM | **D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM dcProbe2RevComp after | ||
**D5: Negative Ctrl (1uM | **D5: Negative Ctrl (1uM dcProbe2RevComp) | ||
===Procedure=== | ===Procedure=== | ||
Line 17: | Line 17: | ||
| 2uM ppMALAT1||1.5 | | 2uM ppMALAT1||1.5 | ||
|- | |- | ||
| 100uM | | 100uM dcProbe2RevComp||3 | ||
|- | |- | ||
| H2O||265.5 | | H2O||265.5 | ||
Line 51: | Line 51: | ||
| Total||100 | | Total||100 | ||
|} | |} | ||
*Aspirate D3&D4 | |||
*Add 100ul 1uM dcProbe2RevComp in 30% formamide + 2X SSC for 15min @ RT | |||
**Preheated to 75C | |||
*Aspirate ALL | |||
*Wash with 2X SSC twice | |||
*Image |
Revision as of 01:44, 18 May 2014
Detecting ppMALAT1 Hybridization
- Use sample from Matt:LabNotes/2014-5-7
- A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2RevComp in one step
- D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM dcProbe2RevComp after
- D5: Negative Ctrl (1uM dcProbe2RevComp)
Procedure
- Add 100ul of ppMALAT1 + dcProbe2-RevComp @45C for 24hrs (6:30p- )
A3-A5
Component | Volume |
10X Ampligase Buffer | 30 |
2uM ppMALAT1 | 1.5 |
100uM dcProbe2RevComp | 3 |
H2O | 265.5 |
Total | 300 |
D3&D4
Component | Volume |
10X Ampligase Buffer | 20 |
2uM ppMALAT1 | 1 |
H2O | 179 |
Total | 200 |
D5
Component | Volume |
10X Ampligase Buffer | 10 |
100uM dcProbe2RevComp | 1 |
H2O | 89 |
Total | 100 |
- Aspirate D3&D4
- Add 100ul 1uM dcProbe2RevComp in 30% formamide + 2X SSC for 15min @ RT
- Preheated to 75C
- Aspirate ALL
- Wash with 2X SSC twice
- Image