Daniel:Notebook/HiResChrPaint/2013-7-25

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Revision as of 19:14, 25 July 2013 by >Djacobse
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S2 Probe Prep (Started 7/23/2013)

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Dye Coupling

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.