Daniel:Notebook/RNAFACS/2015-4-10: Difference between revisions
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##Use TE Buffer | ##Use TE Buffer | ||
#Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight | #Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight | ||
#Continued [[Daniel:Notebook/RNAFACS/2015-4- | #Continued [[Daniel:Notebook/RNAFACS/2015-4-11|tomorrow]] | ||
===Spin Column Results=== | ===Spin Column Results=== | ||
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| width="65" align="center" | ng/uL ssDNA | | width="65" align="center" | ng/uL ssDNA | ||
| width="65" align="center" | pmol/uL dye | | width="65" align="center" | pmol/uL dye | ||
| width=" | | width="85" align="center" | total DNA (ug) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" |
Latest revision as of 23:46, 4 June 2015
In Tube FISH Test[edit]
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 |