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 | *Add 200uL (ACTB+RAB7A+1st rolony probe mix), image --> 2nd round ACTB | ||
*Strip, Add | *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''' | ||
*Strip, Add | |||
====Confocal imaging==== | |||
*20x oil, 16bit, 2048x2048 resolution | |||
*Pick 4 location (discontinued), Z stacking | |||
===Result=== | |||
====RAB7A compare to ACTB==== | |||
*Add three dye probes at one time - Cy5 for 1st rolony, ATTO488 for ACTB, ATTO550 for RAB7A | |||
*RAB7A were detected more than ACTB | |||
{| {{table}} | |||
| 1st Rolonies, Cy5 ([[Media:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_1stRolony.tif|Raw]]) | |||
|| | |||
| ACTB, ATTO488 ([[Media:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_ACTB.tif|Raw]]) | |||
|| | |||
| RAB7A, ATTO550 ([[Media:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_RAB7A.tif|Raw]]) | |||
|- | |||
| [[File:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_1stRolony.png|450px]] | |||
|| | |||
| [[File:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_ACTB.png|450px]] | |||
|| | |||
| [[File:S2_Cy5-1stRolony_ATT488-ACTB_ATTO550-RAB7A_MaxProjection_RAB7A.png|450px]] | |||
| | |||
|} | |||
====Hybridizing and stripping test with imaging 4 different position==== | |||
*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===== | |||
*Each probes annealed again well after stripping...need to analyze more though | |||
*Montage images | |||
**[[Media:Montage1_1stRun-3Dye.png|1st Run - 3 Dyes]] | |||
**[[Media:Montage2_2ndRun-Strip.png|2nd Run - Strip]] | |||
**[[Media:Montage3_3rdRun-3Dye.png|3rd Run - 3 Dyes]] |
Latest revision as of 22:24, 29 October 2013
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