Daniel:Notebook/HiResChrPaint/2013-7-9

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Revision as of 17:38, 10 July 2013 by >Djacobse
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S2 Probe Prep (Started 5/29/2013)[edit]

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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.
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.
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.