Daniel:Notebook/HiResChrPaint/2014-8-21
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Padlock Probes (Started 8/13/2014)
Dye Labeling
Time for dye labeling of the padlock probes. I'm going to label the padlock probes with Alexa 488 and I'm going to make a Cot1-546 for controls.
Sample Matrix
Sample | uL Sample | ug Sample | uL ULS 488 | uL ULS 546 |
Cot1 | 2 | 2 | 0 | 5 |
PPRL | 40 | 1.4 | 2 | 0 |
- 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
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL DNA | dye/probe | bp:dye |
PPRL-488 | 33.5 | 0.6 | 1.0 | 0.6 | 169 |
Cot1-546 | 22.9 | 0.8 | 0.5 | 1.7 | 87 |
The results for PPRL are not very good, but it'll have to do. I should probably add more sample during hybridization.
FISH
Sample Matrix