Daniel:Notebook/HiResChrPaint/2014-3-6
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Overload FISH (Started 3/5/2014)[edit]
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 |
- Following ULYSIS protocol
- Dry samples in vacuum centrifuge
- 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)
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 |