Matt:LabNotes/2015-6-5

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Revision as of 22:43, 5 June 2015 by >Mzcai (→‎Probe Resuspension)
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RNA FISH: Probe Resuspension and Dye Coupling

Probe Resuspension

  • The 5' amino modified (separated by C6) 48-probe set ordered by Dan:
    • GAD1
      • Already resuspended
    • FOXP2
      • Already resuspended
    • CUX2
      • Already resuspended
    • SLC6A1
    • ADARB2
    • SATB2
    • KIT
  • Pick 2-3 genes and resuspend if needed
  1. Resuspend each oligo in 15 uL nuclase-free H2O (1 mM)
  2. Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
    1. Store the remaining oligos at -20C
  3. Add 432 uL nuclase-free, diluting 10:1, for final concentration of 100 uM
    • Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower...
  4. Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy

Dye Coupling

Dan's best practice dye coupling protocol
Generic dye coupling protocol