Matt:LabNotes/2015-6-30: Difference between revisions

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**647/665 (Cy5)
**647/665 (Cy5)
**Filter 4/Filter 6-7
**Filter 4/Filter 6-7
*[[Matt:LabNotes/2015-6-8 | First Try]]
*[[Matt:LabNotes/2015-6-18 | Second Try]]
*[[Matt:LabNotes/2015-6-15 | Probe dye coupling]]
*[[Matt:LabNotes/2015-6-18 | Second batch iPS derived motor neurons from Yeo Lab (Sebastian)]]
**Permeabilized/stored in 70% EtOH at 4C (2 weeks before RNA FISH experiment)
===RNA FISH Procedure===
*Using [[Matt:LabNotes/2015-6-15#Dye_Coupling | CUX2-488 KIT-594 KIT-647]]
*[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]]
*[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]]
*Sample 1 labeled: "RNAFISH1 6.30.2015"
**488
*Sample 2 labeled: "RNAFISH2 6.30.2015"
**594
*Sample 3 labeled: "RNAFISH3 6.30.2015"
**647
#Prepare 15ml Wash Buffer and let sit at RT
#Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
#*Aliquots made by Dan
#Prepare hybridization reaction
#*Hybridization Buffer 100ul
#*Probe 5ul
#Wash the cells with 1ml of PBS (RNase free) 2 times
#Add 1ml Wash Buffer and let sit 10min at RT
#Aspirate
#Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs)
#Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C
#Rinse with 2ml Wash Buffer
#Add 1ml Wash Buffer and incubate 30min at 37C
#Replace with 1ml Wash Buffer with DAPI and incubate 30min at 37C
#Wash with 2X SSC (warmed to 30C) twice
#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 [http://rajlab.seas.upenn.edu/StarSearch/launch.html 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"

Revision as of 23:11, 30 June 2015

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 15ml 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 1ml Wash Buffer and let sit 10min at RT
  6. Aspirate
  7. Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs)
  8. Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C
  9. Rinse with 2ml Wash Buffer
  10. Add 1ml Wash Buffer and incubate 30min at 37C
  11. Replace with 1ml Wash Buffer with DAPI and incubate 30min at 37C
  12. Wash with 2X SSC (warmed to 30C) twice
  13. 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"