Matt:LabNotes/2014-3-4: Difference between revisions
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>Mzcai (Created page with "==3 Genes Secondary Rolonies Mattek Dish== *Hosuk got much more primary rolonies (incl. ACTB and RAB7A) by culturing PGP1F without EGF (Hosuk:LabNotes/2014-3-3) *Since [[M...") |
>Mzcai m (→Procedure) |
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| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| 10X Ampligase||20 | | 10X Ampligase Buffer||20 | ||
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| 200uM ppMALAT1||2 | | 200uM ppMALAT1||2 | ||
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| 200uM ppRAB7A||2 | | 200uM ppRAB7A||2 | ||
|- | |- | ||
| 5U/ul||20 | | 5U/ul Ampligase||20 | ||
|- | |- | ||
| H2O||154 | | H2O||154 |
Revision as of 19:23, 4 March 2014
3 Genes Secondary Rolonies Mattek Dish
- Hosuk got much more primary rolonies (incl. ACTB and RAB7A) by culturing PGP1F without EGF (Hosuk:LabNotes/2014-3-3)
- Since Matt:LabNotes/2014-2-25#Detection resulted in many MALAT1 secondary rolonies but few ACTB or RAB7A
- This is possibly due to sample having very few ACTB or RAB7A
- Also possible the dyes we got from Illumina (dcProbe0-Cy3 and dcProbe1-Cy3) have somehow degraded since MALAT1 detection was using a new dye-probe from IDT
Procedure
- Using Mattek dish from Hosuk:LabNotes/2014-3-3 that was fixed in 4% formalin
- Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min
- Wash with 1X PBS twice
- Anneal Padlock Probes @45C for 24hrs
- 1X Ampligase Buffer
- 2uM of each padlock probe
- 0.5 U/ul Ampligase
Component | Volume |
10X Ampligase Buffer | 20 |
200uM ppMALAT1 | 2 |
200uM ppACTB | 2 |
200uM ppRAB7A | 2 |
5U/ul Ampligase | 20 |
H2O | 154 |
Total | 200 |