Matt:LabNotes/2015-6-30

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Revision as of 23:39, 30 June 2015 by >Mzcai (→‎RNA FISH Procedure)
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Motor Neurons RNA FISH with cooled CCD

  • Photometric QUANTEM Camera
    • Cooled to -30C
    • 100X objective
  • Ex/Em Filters available

File:IMG 0517.JPG

  • Alexa488
    • 495/519 (FITC equivalent)
    • Filter 5/Filter 2
  • Alexa564
    • 556/573 (tetramethylrhodamine)
    • No filters available
  • Alexa594
    • 590/617 (Texas Red)
    • Filter 8/Filter 5
  • Alexa647
    • 647/665 (Cy5)
    • Filter 4/Filter 6-7

RNA FISH Procedure

  • Sample 1 labeled: "RNAFISH1 6.30.2015"
    • 488
  • Sample 2 labeled: "RNAFISH2 6.30.2015"
    • 594
  • Sample 3 labeled: "RNAFISH3 6.30.2015"
    • 647
  1. Prepare 10ml Wash Buffer and let sit at RT
  2. Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
    • Aliquots made by Dan
  3. Prepare hybridization reaction
    • Hybridization Buffer 100ul
    • Probe 5ul
  4. Wash the cells with 1ml of PBS (RNase free) 2 times
  5. Add 2ml Wash Buffer and let sit 10min at RT
  6. Aspirate
  7. Add 105ul hybridization solution to each sample and incubate overnight at 37C (~18hrs)
  8. Prepare 18ml Wash Buffer
  9. Warm up 12ml Wash Buffer and 6ml Wash Buffer with DAPI to 37C
  10. Rinse with 2ml Wash Buffer
  11. Add 2ml Wash Buffer and incubate 30min at 37C
  12. Replace with 2ml Wash Buffer with DAPI and incubate 30min at 37C
    • Try one dish without DAPI?
  13. Wash with 2X SSC (warmed to 30C) twice
  14. Add 2X SSC

Buffer Prep

  • Wash Buffer
    • 20X SSC 5mL
    • Formamide 5mL
    • RNase free H2O 40ml
  • Wash Buffer with DAPI
    • Wash Buffer 10ml
    • DAPI (100ug/ml) 50ul
      • Stored in brown opaque tube in 4C
  • Hybridization Buffer
    • RNAse free water 5.3 mL
    • SSC 20X 1 mL
    • Dextran sulfate 2 mL
    • Formamide 1 mL
    • E coli tRNA 500 uL
    • RVC 200 mM (warm to 37) 100 uL
    • BSA 50 mg/mL 40 uL

Results

  • Olympus Imaging 6/19/2015 (Stored in DARTFISH-RNAFISH folder on desktop)
    • RNAFISH1: Alexa 488 (GFP filter) and Alexa 594 (TXRED filter)
    • RNAFISH2: Alexa 546 (Cy3 filter) and Alexa 647 (Cy5 filter)
  • Use StarSearch from Raj Lab
    • Doesn't work in Chrome browser (use IE)
    • Uses a histogram of pixel intensities and a threshold to determine how many "stars"