Matt:LabNotes/2015-7-23
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RNA FISH[edit]
Dye Probes[edit]
SNAP25_odd-488 & SNAP25_even-594
CUX2-546 (>1month old) & CUX2-647
Procedure[edit]
- 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[edit]
- 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