Daniel:Notebook/RNAFACS/2015-4-10: Difference between revisions

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>Djacobse
(Created page with "=In Tube FISH Test= ==Nuclei Fixation and Dehydration== #Fix cells in 4% paraformaldehyde for 5 min #Centrifuge cells at 1000g for 5 minutes #Wash cells with 70% EtOH #Ethan...")
 
>Djacobse
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=In Tube FISH Test=
=In Tube FISH Test=
[[Daniel:Notebook/RNAFACS|Back to Calendar]]


==Nuclei Fixation and Dehydration==
==Nuclei Fixation and Dehydration==

Revision as of 20:27, 10 April 2015

In Tube FISH Test

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Nuclei Fixation and Dehydration

  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

  • GAD1(48)-488
  • FOXP2(48)-594
  • RBFOX3(24)-546
  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