Daniel:Notebook/RNAFACS/2015-4-8
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In Tube FISH Test[edit]
Time to begin. The Chun lab should be providing me nuclei later today, and I want to begin ASAP.
Buffer Prep[edit]
RPHB
Reagent | Stock Concentration | Final Concentration | Dilution | Volume in 10 mL |
SSC Buffer | 20X | 2X | 1:10 | 1 mL |
Ammonium Sulfate | NA, powder | 2.1 M | NA | 2.775g |
EDTA | 0.5 M | 10 mM | 1:50 | 200 uL |
E. coli tRNA | 20 mg/mL | 1 mg/mL | 1:20 | 500 uL |
BSA | 50 mg/mL | 500 ug/mL | 1:100 | 100 uL |
Formamide | 100% | 25% | 1:04 | 2.5 mL |
nfH2O | - | - | - | Fill to 10mL (~5 mL) |
THESE WILL BE PUSHED BACK TO TOMORROW. DIDN'T GET NUCLEI TODAY.
Nuclei Fixation and Dehydration[edit]
- Fix cells in 4% paraformaldehyde for 5 min
- Centrifuge cells at 1000g for 5 minutes
- Wash cells with 70% EtOH
- Ethanol permeabilization- 70% EtOH overnight at 4C
Dye Labeling[edit]
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
- dye cannot be saved for later use. Use immediately!
- Add 8 uL sample to dye tube
- Incubate in the dark for 1 hour
- Add 10 uL 3M NaOAc and 80 uL nfH20 to sample
- Centri-Sep column purification after incubation
- Use TE Buffer
- Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
- Continued tomorrow