Daniel:Notebook/HiResChrPaint/2013-7-10

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

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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.
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.