Daniel:Notebook/HiResChrPaint/2014-1-14
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Alu Control (Started 1/08/2014)
Dye Coupling Test (with Positive Control)
Repeating the dye coupling test one last time using a positive control as well.
- Following ARES Protocol
Samples
0. Vaccuum centrifuge DNA; resuspend in 2.5 uL nfH2O 1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Add 1.5 uL of sodium bicarbonate buffer 3. Denature samples for 5 minutes at 95C, then snap cool using ice box 4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds *dye cannot be saved for later use. Use immediately! 5. Add 1 uL dye to sample 6. Incubate in the dark for 1 hour 7. Add 20 uL nfH20 to sample; Measure concentration in Nanodrop 8. Centri-Sep column purification 9. Measure concentration in Nanodrop
Labeling Results
Sample | ng/uL dsDNA | pmol/uL dye | pmol/uL ds probe | dye/100bp | ng/uL ssDNA | pmol/uL dye | pmol/uL ss probe | dye/100bp |
Alu2D-Pre Column | 170 | 115 | 113.2 | 116.6 | ||||
Blank-Pre Column | 157.4 | 111 | 103.3 | 110.6 | ||||
Cot1-Pre Column | 150.9 | 105 | 102.1 | 106.4 | ||||
V6S3-Pre Column | 160.3 | 109.2 | 109.6 | 112.9 | ||||
Alu2D-Post Column | 24.4 | 0.5 | 0.4 | 1.4 | 12.6 | 0.5 | 0.4 | 1.3 |
Blank-Post Column | 1 | 0 | 0.0 | 0.0 | 0.2 | 0 | 0.0 | 0.0 |
Cot1-Post Column | 25.9 | 1.7 | 0.2 | 4.3 | 15.6 | 1.3 | 0.3 | 2.8 |
V6S3-Post Column | 41.3 | 0.6 | 0.8 | 1.0 | 24.4 | 0.3 | 0.9 | 0.4 |
This is a big table, but there are a couple of messages to take away from this.
- Excess dye causes a false DNA reading. It doesn't appear to have an effect when dye concentration is low, but when the dye concentration was high (pre-spin column), even the blank sample measured a considerable amount of DNA. I'd recommend always using a blank column and subtracting the background from future samples if pre-purification measurements are needed.
- The Cot1 DNA is reproducibly eluting with dye. Given the other labeling results from previous days (1/13/2014 and 1/10/2014), I'd say that the Cot1 DNA, although having no amine groups, might be being labeled anyway. Or, at least, it's clogging up the column and allowing the dye to co-elute.