Hosuk:LabNotes/2013-10-28

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Revision as of 22:23, 29 October 2013 by >Hosuki78
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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
    • 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

  • 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


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

  • 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