Daniel:Notebook/HiResChrPaint/2013-7-9
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S2 Probe Prep (Started 5/29/2013)[edit]
ULYSIS Dye Coupling (Test 3)[edit]
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[edit]
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.
ULYSIS Dye Coupling (Test 4)[edit]
Based on result from above, I think that using more dye is helpful. However, I also want to see if I can get the same result using less dye and more time.
- Following ULYSIS protocol
1. Add 5 uL DMSO to Alexa dye 488 2. Add 1 uL DNA in 19 uL labeling buffer (Component C) for AmpR5 and AmpR6; 3. Denature DNA for 5 minutes at 95C; snap cool on ice 4. Add 2 uL dye to sample; add 3 uL labeling buffer 5. Incubate AmpR5 for 30 minutes at 80C; incubate AmpR6 for 1 hour, snap cool on ice 6. Purify using Centri-Sep Column
ng/uL DNA | pmol/uL dye | ug DNA in 20 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
AmpR5 (30 min incubation) | 54.5 | 0.5 | 1.1 | 3.3 | 0.2 | 0.3 | 330.3 |
AmpR6 (1 hr incubation) | 62.6 | 0.4 | 1.3 | 3.8 | 0.1 | 0.2 | 474.2 |
I'm suspicious of these results. It's possible that the extra time completely erradicated the dyes, but I find it more likely that there was an error in the process. Post-incubation, the samples have had a faint green color that grows stronger with increasing concentration. This experiment neither showed any hint of a green color, and the pmol/uL values are low enough that I am suspicious they are erroneous.