Daniel:Notebook/HiResChrPaint/2013-7-9
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S2 Probe Prep (Started 5/29/2013)
ULYSIS Dye Coupling
Based on yesterday's results I think I'm going to see what happens if I use all 5 uL of dye available (see ULYSIS protocol) in one labeling. I'll use the oligo Noi gave me again, as I have a lot and it doesn't waste probe.
- Following ULYSIS protocol
1. Add 5 uL DMSO to Alexa dye 488 2. Add 1 uL DNA in 19 uL labeling buffer (Component C) 3. Denature DNA for 5 minutes at 95C; snap cool on ice 4. Add 5 uL dye to sample 5. Incubate for 15 minutes at 80C, snap cool on ice 6. Purify using Centri-Sep Column
Results
ng/uL DNA | pmol/uL dye | ug DNA in 20 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
AmpR4 | 31.8 | 1.4 | 0.636 | 1.9 | 0.7 | 1.45 | 69 |
BLANK | 66.6 | 0.2 | 1.332 | 4.0 | 0.0 | 0.10 | 1009 |
This is not far off from the suggested efficiency values (44-67:1). I think that the efficiency can be improved, however, by also incubating longer.