Daniel:Notebook/HiResChrPaint/2013-7-9

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Revision as of 23:27, 9 July 2013 by >Djacobse
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S2 Probe Prep (Started 5/29/2013)

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ULYSIS Dye Coupling (Test 3)

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

  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)

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