Daniel:Notebook/HiResChrPaint/2014-7-1

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Revision as of 19:04, 1 July 2014 by >Djacobse
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FISH w/ 70% Formamide (Started 06/30/2014)

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Dye Couple

  1. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
    1. For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
    2. 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 column (Centri-Sep recommended)

Centri-Sep Column

  1. Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel
  2. Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle
  3. Drain the column into a wash tube (200-250 uL will drain)
  4. Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid)
  5. Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface
  6. Place column into collection tube and centrifuge at 750xg for 2 minutes
  7. 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