Daniel:Notebook/HiResChrPaint/2013-7-8
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S2 Probe Prep (Started 5/29/2013)[edit]
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.
- Following ULYSIS protocol
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.
- Following ULYSIS protocol
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.