Daniel:Notebook/RNAFISH/2014-12-9

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Revision as of 17:02, 9 December 2014 by >Djacobse (Created page with "=Cell Line RNA FISH= Back to Calendar ==Dye Labeling== Since I am planning on doing RNA FISH using CUX2 and GAD1, and since I would like to use ...")
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Cell Line RNA FISH

Back to Calendar

Dye Labeling

Since I am planning on doing RNA FISH using CUX2 and GAD1, and since I would like to use Alexa 488 and 594, the best dyes I think, I need to do dye labeling. I should be able to use the Alexa dyes, since these are the probes I ordered from IDT.

  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 1 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
  8. Recommend column purification after incubation-Centri-Sep Column