Daniel:Notebook/RNAFACS/2016-1-12
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Poly T Primer Test (Started 01/11/16)[edit]
Buffers[edit]
- Wash Buffer A
- 200 uL Stellaris RNA FISH Wash Buffer A (Biosearch Technologies Cat# SMF-WA1-60)
- Add 700 uL Nuclease-free water
- Add 100 uL Deionized Formamide
- Mix well by vortexing gently
- Wash Buffer A w/DAPI
- 200 uL Stellaris RNA FISH Wash Buffer A (Biosearch Technologies Cat# SMF-WA1-60)
- Add 700 uL Nuclease-free water
- Add 100 uL Deionized Formamide
- Add 1 uL of 20 ug/mL DAPI stock (dilute original 1:1000 and store in 4C)
- Mix well by vortexing gently
Protocol[edit]
- Aspirate out hybridization buffer and add 1 mL of Wash Buffer A
- Incubate in the dark at 37 °C for 30 minutes
- Aspirate the Wash Buffer A, and then add 1 mL of DAPI nuclear stain (Wash Buffer A consisting of 5 ng/mL DAPI) to counterstain the nuclei
- Incubate in the dark at 37 °C for 30 minutes
- Aspirate the DAPI staining buffer, and then add 1 mL of Wash Buffer B. Incubate at room temperature for 2-5 minutes
- Add a small drop (approximately 15 μL) of Vectashield Mounting Medium onto center of dish
- Seal the coverglass perimeter with clear nail polish, and allow to dry
- If necessary, gently wipe away any dried salt off the coverglass with water