Daniel:Notebook/HiResChrPaint/2014-1-14

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Alu Control (Started 1/08/2014)[edit]

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Dye Coupling Test (with Positive Control)[edit]

Repeating the dye coupling test one last time using a positive control as well.

Samples

  • V6S3-A; 13.5 uL, total 1.0 ug
  • Alu2-D; total 1.0 ug
  • Human Cot-1 DNA, 1 ug
0. Vaccuum centrifuge DNA; resuspend in 6 uL nfH2O +3 uL sodium bicarbonate buffer
1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
2. Add 3 uL of sodium bicarbonate buffer
3. Dissolve 1 vial of reactive dye in 4 uL solvent; vortex ≥10 seconds
   *dye cannot be saved for later use.  Use immediately!
4. Add 1 uL dye to each sample
5. Incubate in the dark for 1 hour
6. Add 20 uL nfH20 to sample; 
7. Take 5 uL from sample and dilute with 45 uL nfH2O; Measure concentration in Nanodrop
8. Centri-Sep column purification
9. Measure concentration in Nanodrop

Labeling Results[edit]

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.