Daniel:Notebook/HiResChrPaint/2013-9-5

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BSPP Control (Started 09/03/2013)[edit]

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Dye Coupling[edit]

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 15 uL pmol/uL DNA dye/probe dye/100 bp Base:Dye Ratio (:1)
BSPP 1-546 128.6 4.7 1.9 3.0 1.6 1.2 83
BSPP 2-594 101.6 4.8 1.5 2.4 2.0 1.6 64

Good results, especially BSPP 2. Both of these should work well as controls, and I now have two different options/colors as negative controls.