Daniel:Notebook/RNAFISH/2014-10-11: Difference between revisions
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>Djacobse (Created page with "=ABP Trial Run (Started 10/10/2014)= Back to Calendar ==Dye Labeling-ARES== I need to make the appropriat...") |
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|style="font-weight:bold" height="15" valign="bottom" | Dye | |style="font-weight:bold" height="15" valign="bottom" | Dye | ||
| align="center" valign="bottom" | Alexa- | | align="center" valign="bottom" | Alexa-488 | ||
| align="center" valign="bottom" | Alexa-594 | | align="center" valign="bottom" | Alexa-594 | ||
Revision as of 17:40, 11 October 2014
ABP Trial Run (Started 10/10/2014)
Dye Labeling-ARES
I need to make the appropriate probes, so I'll be labeling with the appropriate ARES dyes.
Sample | FOXP2 | RBFOX3 |
Dye | Alexa-488 | Alexa-594 |
ng/uL | 558 | 568 |
uL added | 10 | 10 |
ug added | 5.6 | 5.7 |
- 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 2 uL dye to sample
- Incubate in the dark for 1 hour
- Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
- Centri Sep purification after incubation
Hybridization
- Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
- Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
- Drop temp to 65C
- Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
- Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
- Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
- Hybridize overnight appropriate temperature
- VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
- important to keep slides in a humid environment