Daniel:Notebook/RNAFACS/2015-4-8

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In Tube FISH Test[edit]

Back to Calendar

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]

  1. Fix cells in 4% paraformaldehyde for 5 min
  2. Centrifuge cells at 1000g for 5 minutes
  3. Wash cells with 70% EtOH
  4. Ethanol permeabilization- 70% EtOH overnight at 4C

Dye Labeling[edit]

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 8 uL sample to dye tube
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL nfH20 to sample
  8. Centri-Sep column purification after incubation
    1. Use TE Buffer
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
  10. Continued tomorrow