Daniel:Notebook/RNAFACS/2015-4-10
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In Tube FISH Test[edit]
Back to Calendar
Nuclei Fixation and Dehydration[edit]
- Fix cells in 4% paraformaldehyde for 5 min
- Centrifuge cells at 1000g for 5 minutes
- Wash cells with 70% EtOH
- Ethanol permeabilization- 70% EtOH overnight at 4C
Dye Labeling[edit]
- GAD1(48)-488
- FOXP2(48)-594
- RBFOX3(24)-546
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 5 uL DNA 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 solvent; vortex ≥10 seconds
- 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 nfH20 to sample
- Centri-Sep column purification after incubation
- Use TE Buffer
- Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
- Continued tomorrow
Spin Column Results[edit]
Probe
|
ng/uL ssDNA
|
pmol/uL dye
|
total DNA (ug)
|
GAD1-488
|
22.3
|
4.4
|
2.23
|
RBFOX3-546
|
21.0
|
13.0
|
2.1
|
FOXP2-594
|
48.5
|
33.5
|
4.85
|