Daniel:Notebook/HiResChrPaint/2013-7-25
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S2 Probe Prep (Started 7/23/2013)
Dye Coupling
- Following ARES protocol
0. Dry samples in a vaccuum centrifuge
For each
1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Resuspend DNA in 5 uL nfH20 3. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer in PCR tube 4. Denature samples for 5 minutes at 95C, then snap cool using ice box 5. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds *dye cannot be saved for later use. Use immediately! 6. Add 2 uL dye to sample 7. Incubate in the dark for 1 hour 8. Add 10 uL nfH20 to sample 9. Purify using Centri Sep column 10. Measure concentrations using Microarray setting in nanodrop
Labeling Results
ng/uL DNA | pmol/uL dye | ug DNA in 15 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
V4S3 | 106.6 | 3.8 | 1.6 | 4.0 | 1.0 | 1.2 | 85 |
V6S3 | 133.8 | 5.5 | 2.0 | 5.0 | 1.1 | 1.4 | 74 |
Again, the labeling results weren't fantastic, but they should serve as workable probes for FISH experiments.