Daniel:Notebook/RNAFACS/2015-4-10: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 28: | Line 28: | ||
#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/2015-4-9|tomorrow]] | #Continued [[Daniel:Notebook/RNAFACS/2015-4-9|tomorrow]] | ||
===Spin Column Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#CCC0DA;font-size:12pt" | |||
| width="96" height="30" valign="bottom" | Probe | |||
| width="65" align="center" | ng/uL ssDNA | |||
| width="65" align="center" | pmol/uL dye | |||
| width="65" align="center" | total DNA (ug) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | GAD1-488 | |||
| align="center" align="center" valign="bottom" | 22.3 | |||
| align="center" align="center" valign="bottom" | 4.4 | |||
| align="center" align="center" valign="bottom" | 2.23 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | RBFOX3-546 | |||
| align="center" align="center" valign="bottom" | 21.0 | |||
| align="center" align="center" valign="bottom" | 13.0 | |||
| align="center" align="center" valign="bottom" | 2.1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | FOXP2-594 | |||
| align="center" align="center" valign="bottom" | 48.5 | |||
| align="center" align="center" valign="bottom" | 33.5 | |||
| align="center" align="center" valign="bottom" | 4.85 | |||
|} |
Revision as of 20:31, 11 April 2015
In Tube FISH Test
Nuclei Fixation and Dehydration
- 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
- 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
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 |