Daniel:Notebook/HiResChrPaint/2013-7-3
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S2 Probe Prep (Started 5/29/2013)[edit]
ULYSIS Dye Coupling[edit]
Trying the ULYSIS dye labeling technique using V4S2 sample without aadUTP as a trial run. Sample has been dried in a vacuum centrifuge and will need to be resuspended.
V4S2 (no aa-dUTP) ~1.5 ug
- Following ULYSIS protocol
1. Add 5 uL DMSO to Alexa dye 488 2. Resuspend DNA in 20 uL labeling buffer (Component C) 3. Denature DNA for 5 minutes at 95C; snap cool on ice 4. Add 1 uL dye to sample; add 4 uL labeling buffer 5. Incubate for 15 minutes at 80C, snap cool on ice 6. Purify using Centri-Sep Column
Results[edit]
ng/uL DNA | ug DNA in 20 uL | pmol/uL DNA | pmol/uL dye | dye/probe | dye/100bp |
111.9 | 2.2 | 4.2 | 1.9 | 0.5 | 0.62 |
Seems like this kit doesn't work as well as the ARES. The instructions say labeling efficiency should be around 44 fluorophores/67 bases (guanine only, actual labeling efficiency the way I calculate it should be around 16.4% assuming equal base probability). I'm going to look into possible reasons why this ratio is so low for me.