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 : Resuspension --> 200uM
*RAB7A --> ATTO550 laeled
*1uL 200uM RAB7A + 2uL Cy5 1st rolony adapter in 197 uL 2x SSC
**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
*1uL 200uM ACTB + 2uL Cy5 1st rolony adapter in 197 uL 2x SSC --> 2 tubes
*2mL 80% Formamide 2x SSC, PBS, Aspiration setup, vacuum pump
*1uL 200uM RAB7A + 2uL Cy5 1st rolony adapter in 197 uL 2x SSC --> 2 tubes
*2mL 80% Formamide
*2x SSC, PBS
*2x SSC, PBS
*Aspiration setup, vacuum pump
*Aspiration setup, vacuum pump
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====Procedure====
====Procedure====
*Strip, Add 100uL (RAB7A+1st rolony probe mix), image --> 1st round RAB7A
*Strip, Add 200uL (ACTB+RAB7A+1st rolony probe mix), image --> 1st round RAB7A
*Strip, Add 100uL (ACTB+1st rolony probe mix), image --> 1st round ACTB
*Strip, imaging
*Strip, Add 100uL (RAB7A+1st rolony probe mix), image --> 2nd round RAB7A
*Add 200uL (ACTB+RAB7A+1st rolony probe mix), image --> 2nd round ACTB
*Strip, Add 100uL (ACTB+1st rolony probe mix), image --> 2nd round ACTB
*Strip, imaging
*Strip, Add 100uL (RAB7A only), image --> 1st round RAB7A only
**Add 200uL (ACTB+1st only), image --> 1st round ACTB only '''-->done at 10/29'''
*Strip, Add 100uL (ACTB only), image --> 1st round ACTB only
**Strip, imaging
*Strip, Add 100uL (RAB7A only), image --> 2nd round RAB7A only
**Add 200uL (RAB7A+1st only), image --> 1st round RAB7A only '''-->not done yet'''
*Strip, Add 100uL (ACTB only), image --> 2nd round ACTB only
 
 
====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