Hosuk:LabNotes/2013-10-28
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Detect RAB7A, and compare to ACTB[edit]
- Use 10/22 sample (MatTek dish, S2)
- RAB7A --> ATTO550 laeled
- Resuspension --> 200uM
Preparation[edit]
- 1uL 200uM ACTB + 1uL 200uM RAB7A + 2uM Cy5 100uM 1st rolony adapter in 196 uL 2x SSC --> 2 tubes
- 1uL 200uM ACTB in 199 uL 2x SSC --> 2 tubes
- 1uL 200uM RAB7A + 199 uL 2x SSC --> 2 tubes
- 2mL 80% Formamide 2x SSC, PBS, Aspiration setup, vacuum pump
- 2x SSC, PBS
- Aspiration setup, vacuum pump
Procedure[edit]
- Strip, Add 200uL (ACTB+RAB7A+1st rolony probe mix), image --> 1st round RAB7A
- Strip, imaging
- Add 200uL (ACTB+RAB7A+1st rolony probe mix), image --> 2nd round ACTB
- Strip, imaging
- Add 200uL (ACTB+1st only), image --> 1st round ACTB only -->done at 10/29
- Strip, imaging
- Add 200uL (RAB7A+1st only), image --> 1st round RAB7A only -->not done yet
Confocal imaging[edit]
- 20x oil, 16bit, 2048x2048 resolution
- Pick 4 location (discontinued), Z stacking
Result[edit]
RAB7A compare to ACTB[edit]
- Add three dye probes at one time - Cy5 for 1st rolony, ATTO488 for ACTB, ATTO550 for RAB7A
- RAB7A were detected more than ACTB
1st Rolonies, Cy5 (Raw) | ACTB, ATTO488 (Raw) | RAB7A, ATTO550 (Raw) | |||
File:S2 Cy5-1stRolony ATT488-ACTB ATTO550-RAB7A MaxProjection 1stRolony.png | File:S2 Cy5-1stRolony ATT488-ACTB ATTO550-RAB7A MaxProjection ACTB.png | File:S2 Cy5-1stRolony ATT488-ACTB ATTO550-RAB7A MaxProjection RAB7A.png |
Hybridizing and stripping test with imaging 4 different position[edit]
- Add 3 dye probes (1st rolony, ACTB, and RAB7A), imaging at 4 positions
- Strip, and image again
- Add 3 dye probes (1st rolony, ACTB, and RAB7A), imaging at 4 positions
Result[edit]
- Each probes annealed again well after stripping...need to analyze more though
- Montage images