Hosuk:LabNotes/2013-10-28: Difference between revisions
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===Detect RAB7A, and compare to ACTB=== | ===Detect RAB7A, and compare to ACTB=== | ||
*Use 10/22 sample (MatTek dish, S2) | *Use 10/22 sample (MatTek dish, S2) | ||
*RAB7A | *RAB7A --> ATTO550 laeled | ||
* | **Resuspension --> 200uM | ||
====Preparation==== | ====Preparation==== | ||
*1uL 200uM ACTB + 199 uL 2x SSC --> 2 tubes | *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 | *1uL 200uM RAB7A + 199 uL 2x SSC --> 2 tubes | ||
*2mL 80% Formamide 2x SSC, PBS, Aspiration setup, vacuum pump | |||
*2mL 80% Formamide | |||
*2x SSC, PBS | *2x SSC, PBS | ||
*Aspiration setup, vacuum pump | *Aspiration setup, vacuum pump | ||
Line 19: | Line 18: | ||
====Procedure==== | ====Procedure==== | ||
*Strip, Add | *Strip, Add 200uL (ACTB+RAB7A+1st rolony probe mix), image --> 1st round RAB7A | ||
*Strip, Add | *Strip, imaging | ||
*Strip, | *Add 200uL (ACTB+RAB7A+1st rolony probe mix), image --> 2nd round ACTB | ||
* | *Strip, imaging | ||
* | |||
*Strip, Add | |||
*Strip, Add | *optional experiment | ||
*Strip, Add | **Add 200uL (RAB7A only), image --> 1st round RAB7A only | ||
**Strip, imaging | |||
**Add 200uL (ACTB+1st only), image --> 1st round ACTB only | |||
**Strip, imaging | |||
**Add 200uL (RAB7A+1st only), image --> 2nd round RAB7A only | |||
**Strip, imaging | |||
**Add 200uL (ACTB+1st only), image --> 2nd round ACTB only | |||
====Confocal imaging==== | |||
*20x oil, 16bit, 2048x2048 resolution | |||
*Pick 4 location (discontinued), Z stacking |
Revision as of 23:09, 28 October 2013
Detect RAB7A, and compare to ACTB
- Use 10/22 sample (MatTek dish, S2)
- RAB7A --> ATTO550 laeled
- Resuspension --> 200uM
Preparation
- 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
- 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
- optional experiment
- Add 200uL (RAB7A only), image --> 1st round RAB7A only
- Strip, imaging
- Add 200uL (ACTB+1st only), image --> 1st round ACTB only
- Strip, imaging
- Add 200uL (RAB7A+1st only), image --> 2nd round RAB7A only
- Strip, imaging
- Add 200uL (ACTB+1st only), image --> 2nd round ACTB only
Confocal imaging
- 20x oil, 16bit, 2048x2048 resolution
- Pick 4 location (discontinued), Z stacking