Daniel:Notebook/HiResChrPaint/2013-10-25

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Revision as of 22:55, 25 October 2013 by >Djacobse (→‎Labeling Results)
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BSPP Control (Started 10/22/2013)[edit]

Back to Calendar

Dye Coupling[edit]

Just like with 9/03/2013 I'll be coupling BSPP-1 to Alexa 546 and BSPP-2 to Alexa 594

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.