Matt:LabNotes/2014-3-4
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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 (Put in at noon, some bubbles and hard to keep on middle glass)
- 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 |