Daniel:Notebook/HiResChrPaint/2014-8-21
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Padlock Probes (Started 8/13/2014)[edit]
Dye Labeling[edit]
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[edit]
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[edit]
Sample Matrix
Sample | uL PPRL-488 (pmol) | uL Cot1-546 (pmol) | uL DMD-546 (pmol) | uL Cot1-488 (ug) |
A | 25 (25) | 25 (0.6) | 0 | 0 |
B | 0 | 0 | 10.5 (40) | 6 (0.3) |
- Allow stored samples to warm to room temperature
- Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
- Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
- Remove samples and allow to cool to room temperature
- Add 25 uL hybridization cocktail to center
- Add 22x22 #1.5 coverslip; seal with rubber cement
- Allow the rubber cement to air-dry for 5 minutes at room temperature
- Denature for 2.5 minutes at 92C; keep humid
- Transfer slides to a humidified chamber and hybridize overnight at 37C