Daniel:Notebook/HiResChrPaint/2013-10-25
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BSPP Control (Started 10/22/2013)[edit]
Dye Coupling[edit]
Just like with 9/03/2013 I'll be coupling BSPP-1 to Alexa 546 and BSPP-2 to Alexa 594
- 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[edit]
ng/uL DNA | pmol/uL dye | ug DNA in 20 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
BSPP 1-546 | 52 | 1.7 | 1.0 | 1.9 | 0.9 | 1.1 | 93 |
BSPP 2-594 | 68.8 | 2.6 | 1.4 | 2.6 | 1.0 | 1.2 | 80 |
So not great, but overall pretty good. Unfortunately there is just not a lot of either (1.9 and 2.6 pmol/uL), but they'll last 4 or 5 FISH experiments.