Daniel:Notebook/HiResChrPaint/2014-3-6

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Overload FISH (Started 3/5/2014)[edit]

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Dye Labeling[edit]

Samples:

Sample Amount Dye
V6S2(A+B) 6.3 ug 488
V6S3(A+B) 6.9 ug 546
Cot1 6uL = 6ug 488
BSPP1B 2.7 ug 546
  1. Dry samples in vacuum centrifuge
  2. 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!
  3. Resuspend DNA in 20 uL labeling buffer (Component C)
  4. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
  5. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
  6. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
  7. Purify using a column (Centri-Sep recommended)
File:ULYSIS ReactionTable.png

Labeling Results[edit]

Sample ng/uL dsDNA pmol/uL dye Length (bp) pmol/uL probe dye/probe bp:dye
V6S2 260.9 2.3 81 4.9 0.5 172
Cot1 137.5 2.9 400 0.5 5.6 15
V6S3 66.1 1.7 81 1.2 1.4 59
BSPP1 83.9 0.8 100 1.3 0.6 129