Daniel:Notebook/RNAFISH/2014-10-11
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ABP Trial Run (Started 10/10/2014)[edit]
Dye Labeling-ARES[edit]
I need to make the appropriate probes, so I'll be labeling with the appropriate ARES dyes.
Sample | FOXP2 | RBFOX3 |
Dye | Alexa-594 | Alexa-488 |
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
Labeling Results[edit]
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye:probe | bp:dye |
FOXP2-594 | 61.3 | 11.9 | 9.3 | 1.3 | 16 |
RBFOX3-488 | 54.9 | 9 | 8.3 | 1.1 | 18 |
As usual wit ARES, looks great.
Hybridization[edit]
- 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
Buffers[edit]
- Prehybridization solution:
- 50% deionized formamide
- 50% 2xSSC pH 7.0
- Add dextran sulfate to 1%
- Hybridization solution A-Long Cai
- 2X SSC
- 20% Formamide
- 10% Dextran Sulfate
- Hybridization solution B-Arjun Raj
- 2X SSC
- 10% Formamide
- 10% Dextran Sulfate
- 0.02% RNAse-free BSA
- 50 μg E.coli tRNA