Matt:LabNotes/2015-6-15
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Motor Neurons RNA FISH Dye Coupling[edit]
- Previous RNA FISH attempt detected no signal
- Try again with higher expressed genes (CUX2 and KIT)
Probe Resuspension[edit]
- Table of ordered plates
- KIT: Plate=KIT-SNAP25-20150128 Wells=A1-D12
- SNAP25: Plate=KIT-SNAP25-20150128 Wells=E1-H12
- Table of ordered plates
- Resuspend each oligo in 15 uL nuclase-free H2O (1 mM)
- Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
- Store the remaining oligos at -20C
- 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
- 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
- Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
- Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- 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!
- Add 8 uL sample to dye tube
- Incubate in the dark for 1 hour
- Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
- Centri-Sep column purification after incubation
- Use 1X TE Buffer
- No vacufuge
- Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
- Centrifuge at 4 C for 30 minutes (14,000 rpm)
- Remove supernatant and add 750 uL chilled 75% EtOH
- Centrifuge 5 minutes at 4 C
- Dry the pellet in the hood
- Resuspend pellet in 12 uL TE
- 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 |