Daniel:Notebook/HiResChrPaint/2014-1-23
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Alu Control (Started 01/08/2014)
Dye Coupling
1. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C) *For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C) ***VORTEX THESE SOLUTIONS VIGOROUSLY BEFORE USE EVERY TIME! 2. Resuspend DNA in 20 uL labeling buffer (Component C) 3. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly 4. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL 5. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath 6. Purify using a Centri-Sep column
Labeling Results
Pre Column | Post Column | |||||||||
Sample | ng/uL dsDNA | pmol/uL dye | pmol/uL ss probe | base:dye | ng/uL ssDNA | pmol/uL dye | pmol/uL ss probe | base:dye | DNA retention (%) | Dye retention (%) |
Cot1-A | 0 | 0.9 | 0.0 | 0.0 | 3.3 | 0.3 | 0.1 | 33.3 | NA | 2.22 |
Cot1-B | 1.4 | 0.8 | 0.0 | 5.3 | 13.1 | 0.3 | 0.2 | 132.3 | 62.38 | 2.50 |
Blank | 0 | 0.8 | 0.0 | 0.0 | 0 | 0 | 0.0 | NA | NA | 0.00 |
DNA Only | 2.1 | 0 | 0.0 | NA | 19.4 | 0 | 0.3 | NA | 61.59 | NA |
So these are pretty expected results, at least from the controls. The DNA only sample showed similar DNA retention to the mixed sample (but with no dye retention of course). The blank sample, which had dye but no DNA, had no DNA retention and no dye retention, indicating that the ULS dye did not move quickly through the column. The samples themselves, however, are harder to piece out. One problem is that the pre-column samples were diluted 1:10 before being measured in the nanodrop.