Matt:LabNotes/2015-6-15

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Motor Neurons RNA FISH Dye Coupling[edit]

Probe Resuspension[edit]

  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 450 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...
    • Goal is to get 1ug/ul DNA concentration
  4. Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
' measured (ng/ul) normalized (ng/ul) Average Stdev
KIT 1:1 1053.5 1053.5
KIT 1:5 212.5 1062.5
KIT 1:10 104.9 1049 1055 6.87
SNAP25 1:1 1039.5 1039.5
SNAP25 1:5 207.7 1038.5
SNAP25 1:10 107.4 1074 1050.67 20.21

Dye Coupling[edit]

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

  • Sebastian said in email the two highest expressed genes from our 48 probe set of genes are CUX2 and KIT
  • I am going to try both 488/594 and 546/647 pairs of fluorophores in case one set is not compatible with our Olympus filter cubes

CUX2-488 KIT-594 CUX2-546 KIT-647

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 5 uL DNA (~1ug/ul) 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 DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
    • 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 1xTE to sample
  8. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
    • No vacufuge
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
  10. Centrifuge at 4 C for 30 minutes (14,000 rpm)
  11. Remove supernatant and add 750 uL chilled 75% EtOH
  12. Centrifuge 5 minutes at 4 C
  13. Dry the pellet in the hood
  14. Resuspend pellet in 12 uL TE
  15. Check dye:probe ratio with Nanodrop
    • Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer)
    • Starting DNA mass = 5ul * 1ug/ul
Probe ssDNA (ng/ul) 488 dye (pmol/ul) 594 dye (pmol/ul) 546 dye (pmol/ul) 647 dye (pmol/ul) dye:probe ratio ssDNA mass (ug) ssDNA start mass (ug) ssDNA Yield (%)
CUX2-488 227.7 39.1 0 N/A N/A 1.11 2.7324 5 54.648
KIT-594 251.2 -0.6 51.6 N/A N/A 1.33 3.0144 5 60.288
CUX2-546 246.9 N/A N/A 43.7 0.2 1.15 2.9628 5 59.256
KIT-647 268.6 N/A N/A 6.8 45.6 1.10 3.2232 5 64.464