Matt:LabNotes/2015-6-5

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RNA FISH: Probe Resuspension and Dye Coupling[edit]

Probe Resuspension[edit]

  • 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 350 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 and this is what Dan has found to get closest to that
  4. Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
' measured (ng/ul) normalized (ng/ul) Average Stdev
ADARB2 1:1 1228 1228
ADARB2 1:5 271.8 1359
ADARB2 1:10 135.4 1354 1313.7 74.2
CUX2 1:1 1227.6 1227.6
CUX2 1:5 258.5 1292.5
CUX2 1:10 128.8 1288 1269.4 36.2
SATB2 1:1 1328.8 1328.8
SATB2 1:5 290 1450
SATB2 1:10 138.2 1382 1386.9 60.8
SLC6A1 1:1 1356.8 1356.8
SLC6A1 1:5 300.3 1501.5
SLC6A1 1:10 147.4 1474 1444.1 76.8
  • For ADARB2 & CUX2 with approx 1300ng/ul
    • Add 132ul H2O to 440ul
    • 1300ng/ul x 440ul = 1000ng/ul x 572ul -> 572-440=132ul H2O added
  • For SATB2 & SLC6A1 with approx 1400ng/ul
    • Add 176ul H2O to 440ul
    • 1400ng/ul x 440ul = 1000ng/ul x 616ul -> 616-440=176ul H2O added

Dye Coupling[edit]

Dan's best practice dye coupling protocol
Generic dye coupling protocol SLC6A1-488 SATB2-594

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O 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 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
  1. Centrifuge at 4 C for 30 minutes (14,000 rpm)
  2. Remove supernatant and add 750 uL chilled 75% EtOH
  3. Centrifuge 5 minutes at 4 C
  4. Dry the pellet in the hood
  5. Resuspend pellet in 12 uL TE
  6. 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 ssDNA pmol/ul dye:probe ratio ssDNA mass (ug) ssDNA start mass (ug) ssDNA Yield (%)
SLC6A1-488 247.8 43.3 0.6 38.18 1.13 2.97 5 59.4
SATB2-594 260.4 -0.6 52.6 40.12 1.31 3.12 5 62.4