Daniel:Notebook/HiResChrPaint/2013-7-15: Difference between revisions

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Great results from ARES.  Good but not great from ULS.
Pretty good results from ARES.  Good but not great from ULS.  Both slightly higher than the 44-67 bp/dye that the ULS argues is best.  I haven't seen data on what ARES ratios are best, but the [[Daniel:Notebook/HiResChrPaint/2013-7-12|results]] for ULS indicate that one fluorophore per 50 bp is around the optimal labeling ratio for visualization.  Still, I think that these might be good enough to try FISH.

Revision as of 19:51, 15 July 2013

S2 Probe Prep (Started 5/29/2013)

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ARES Labeling of V6S2

Trying ARES labeling of V6S2 set randomly primed with aa-dUTP.

V6S2 ~ 1.2 ug DNA

1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
2. Resuspend DNA in 2.5uL 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 11 uL nfH20 to sample
9. Purify using Centri Sep column

ARES Results

  ng/uL DNA pmol/uL dye ug DNA in 15 uL pmol/uL DNA dye/probe dye/100 bp Base:Dye Ratio (:1)
V6S2 189.1 7.9 2.8 7.1 1.1 1.4 73

ULYSIS Labeling of V6S2

Trying ULS labeling of V6S2 set randomly primed with aa-dUTP.

V6S2 ~ 1.5 ug DNA

1. Resuspend DNA in 20 uL labeling buffer (Component C)
2. Denature DNA for 5 minutes at 95C; snap cool on ice
3. Add 1 uL dye to sample
4. Incubate for 20 minutes at 80C, snap cool on ice
5. Purify using Centri Sep column

ULYSIS Results

  ng/uL DNA pmol/uL dye ug DNA in 15 uL pmol/uL DNA dye/probe dye/100 bp Base:Dye Ratio (:1)
V6S2 237 6.1 3.6 8.9 0.7 0.8 118

Pretty good results from ARES. Good but not great from ULS. Both slightly higher than the 44-67 bp/dye that the ULS argues is best. I haven't seen data on what ARES ratios are best, but the results for ULS indicate that one fluorophore per 50 bp is around the optimal labeling ratio for visualization. Still, I think that these might be good enough to try FISH.