Matt:LabNotes/2015-6-18: Difference between revisions
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**Permeabilized/store in 70% EtOH at 4C (~48 hours before RNA FISH experiment) | **Permeabilized/store in 70% EtOH at 4C (~48 hours before RNA FISH experiment) | ||
===RNA FISH=== | ===RNA FISH Procedure=== | ||
*Using [[Matt:LabNotes/2015-6-15#Dye_Coupling | CUX2-488 KIT-594 CUX2-546 KIT-647]] | *Using [[Matt:LabNotes/2015-6-15#Dye_Coupling | CUX2-488 KIT-594 CUX2-546 KIT-647]] | ||
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**RVC 200 mM (warm to 37) 100 uL | **RVC 200 mM (warm to 37) 100 uL | ||
**BSA 50 mg/mL 40 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) |
Revision as of 01:22, 25 June 2015
Motor Neurons RNA FISH
- Picked up second batch iPS derived motor neurons from Yeo Lab (Sebastian)
- Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
- RT for 15min
- Permeabilized/store in 70% EtOH at 4C (~48 hours before RNA FISH experiment)
- Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
RNA FISH Procedure
- Sample 1 labeled: "RNAFISH1 6.18.2015"
- 488 and 594
- Sample 2 labeled: "RNAFISH2 6.18.2015"
- 546 and 647
- Prepare 10ml Wash Buffer and let sit at RT
- Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
- Aliquots made by Dan
- Prepare hybridization reaction (2X volume)
- Hybridization Buffer 200ul
- Probe 10ul of each (5ul-488+5ul-594 and 5ul-546+5ul-647)
- 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 StarSearch from Raj Lab
- Doesn't work in Chrome browser (use IE)