Daniel:Notebook/HiResChrPaint/2013-7-10
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S2 Probe Prep (Started 5/29/2013)[edit]
Summary of ULYSIS Tests using AmpR Oligo[edit]
Sample | uL dye Added | Incubation Time (min) | pmol/uL DNA | pmol/uL dye | dye/probe | base:dye ratio |
AmpR1 | 1 | 15 | 3.5 | 1.2 | 0.341 | 147 |
AmpR2 | 1 | 15 | 3.1 | 1.2 | 0.384 | 130 |
AmpR3 | 2 | 15 | 3.4 | 1.9 | 0.552 | 91 |
AmpR4 | 5 | 15 | 1.9 | 1.4 | 0.7 | 69 |
AmpR5 | 2 | 30 | 3.3 | 0.5 | 0.2 | 330 |
AmpR6 | 2 | 60 | 3.8 | 0.4 | 0.1 | 474 |
BLANK | 0 | 15 | 4 | 0.2 | 0 | 1009 |
ULYSIS Dye Coupling (Test 5)[edit]
Based on yesterday's results I want to retry the time experiment, this time only with 20 minutes instead of 30 or 60. Also, I'm going to use 1 sample of the dye I made yesterday (AmpR7) and one with a "fresh" dye I'll prepare today (AmpR8). Hopefully this will explain the weird results from yesterday. Also, I'm going to do a pre/post comparison for the column, to see what that yields.
- 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 1 uL dye to sample; add 4 uL labeling buffer 5. Incubate for 20 minutes at 80C, snap cool on ice 6. Measure concentrations in Nanodrop 7. Purify using Centri-Sep Column
Results[edit]
Labeling Efficiency
ng/uL DNA | pmol/uL dye | ug DNA in 20 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
AmpR7 | 42.3 | 2.3 | 0.8 | 2.6 | 0.9 | 1.8 | 56 |
AmpR8 | 59.9 | 0.7 | 1.2 | 3.6 | 0.2 | 0.4 | 259 |
Column Efficiency
Pre ng/uL DNA | Pre pmol/uL dye | ng/uL DNA | pmol/uL dye | Column Yield (% DNA) | Column Yield (% dye) | |
AmpR7 | 61.4 | 15.4 | 42.3 | 2.3 | 59.9 | 13.0 |
AmpR8 | 60.8 | 2.8 | 59.9 | 0.7 | 85.7 | 21.7 |
Not really sure what happened here. First I'll note that I couldn't get a full uL of dye from the old tube, so I actually got better results than ever from <1 uL dye. The results would suggest I mixed up the tubes, since I would have expected the dye made today to perform better.