Daniel:Notebook/HiResChrPaint/2013-7-15

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S2 Probe Prep (Started 5/29/2013)[edit]

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ARES Labeling of V6S2[edit]

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[edit]

  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[edit]

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[edit]

  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

Discussion[edit]

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. Looking at the results, it would seem either the TBU gel greatly underestimated the concentration of DNA left or the nanodrop greatly overestimated the concentration. Probably the first. It would however, make sense that the ULS did not have a great yield, since by these results it would seem I have more DNA than I added dye for.

Grant Proposal[edit]

Dr. Zhang has been having me write a small collaborative grant with Molecular Probe. After several rounds of revision we have a version deemed okay to send to our collaborators. It is attached here: File:MolecularProbeGrant.docx