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
 
*Strip, Add 100uL (ACTB only), image --> 1st round ACTB only
 
*Strip, Add 100uL (RAB7A only), image --> 2nd round RAB7A only
*optional experiment
*Strip, Add 100uL (ACTB only), image --> 2nd round ACTB only
**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