Daniel:Notebook/HiResChrPaint/2014-7-1
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FISH w/ 70% Formamide (Started 06/30/2014)
Dye Couple
- 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!
- Resuspend DNA in 20 uL labeling buffer (Component C)
- Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
- Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
- Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
- Purify using a column (Centri-Sep recommended)
Centri-Sep Column
- Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel
- Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle
- Drain the column into a wash tube (200-250 uL will drain)
- Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid)
- Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface
- Place column into collection tube and centrifuge at 750xg for 2 minutes
- Dry sample in vacuum centrifuge
Labeling Results
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye/probe | bp:dye ratio |
V6S2-546 | 54.4 | 2.2 | 1.6 | 1.4 | 75 |
V6S3-488 | 163.5 | 0.7 | 4.9 | 0.1 | 708 |
Cot1-488 | 50.3 | 1 | 0.5 | 2.0 | 152 |