Daniel:Notebook/HiResChrPaint/2013-7-8

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

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ULYSIS Dye Coupling (Test)[edit]

Going to use the probe Noi gave me a while back to test yield of ULYSIS dye coupling.  Going to try two for better confidence.
1. Add 1 uL DNA in 19 uL labeling buffer (Component C)
2. Denature DNA for 5 minutes at 95C; snap cool on ice
3. Add 1 uL dye to sample; add 4 uL labeling buffer
4. Incubate for 15 minutes at 80C, snap cool on ice
5. Purify using Centri-Sep Column

Results[edit]

  AmpR AmpR 1 AmpR 2
ng/uL DNA 538 58.1 51.6
pmol/uL dye 0.1 1.2 1.2
ug DNA 0.5 1.2 1.0
pmol/uL DNA 32.6 3.5 3.1
dye/probe 0.003 0.341 0.384
dye/100bp 0.0 0.7 0.8
Base:dye ratio NA 146.6 130.2

ULYSIS Dye Coupling (Test 2)[edit]

Following a recommendation from Matt, I'm going to use more dye (go figure, right?). If this reaction shows increased yield to the original
method, I'll scale up following trials by even more.  Still using the oligo Noi gave me as for the trial run.
1. Add 1 uL DNA in 19 uL labeling buffer (Component C)
2. Denature DNA for 5 minutes at 95C; snap cool on ice
3. Add 2 uL dye to sample; add 3 uL labeling buffer
4. Incubate for 15 minutes at 80C, snap cool on ice
5. Purify using Centri-Sep Column

Results[edit]

  AmpR3 BLANK
ng/uL DNA 56.8 55.7
pmol/uL dye 1.9 0.1
ug DNA in 20 uL 1.1 1.1
pmol/uL DNA 3.4 3.4
dye/probe 0.552 0.030
dye/100bp 1.1 0.1
Base:Dye Ratio (:1) 90.6 NA
It would seem that by doubling the dye used I got a 1.4x increase in labeling.  For the next trial I will try using 5x the recommended amount
of dye (a full tube).  This will hopefully give me the labeling efficiency I desire.