Daniel:Notebook/HiResChrPaint/2013-7-3

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

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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

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.