Daniel:Notebook/HiResChrPaint/2013-7-15: Difference between revisions
Jump to navigation
Jump to search
>Djacobse No edit summary |
>Djacobse No edit summary |
||
Line 86: | Line 86: | ||
|} | |} | ||
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)
ARES Labeling of V6S2
Trying ARES labeling of V6S2 set randomly primed with aa-dUTP.
V6S2 ~ 1.2 ug DNA
- Following ARES Protocol
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
- Following ULYSIS Protocol
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.