Daniel:Notebook/RNAFISH/2015-3-5
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Linnarsson Lab Protocol 2 (Started 3/4/2015)[edit]
Dye Coupling (Continued; Ethanol Precipitation)[edit]
- Remove from -80C
- Centrifuge at 4 C at 12000 rpm for 30 minutes
- Remove supernatant and add 700 uL chilled 75% EtOH
- Centrifuge at minutes at 4 C for 10 minutes at 12000 rpm
- Remove supernatant and dry the pellet in the hood
- Resuspend pellet in 7.5 uL TE buffer
- Store DNA at 4C for immediate use
Nanodrop Results[edit]
ng/uL ssDNA | pmol/uL dye | ng/uL ssDNA | pmol/uL dye | dye/probe | bp:dye ratio | EtOH Yield (%) DNA | EtOH Yield Dye (%) | Total Yield DNA (%) | uM Labeled DNA | |
FOXP2-488 | 23.5 | 6.7 | 230.3 | 40.0 | 1.1 | 17 | 73.50 | 44.78 | 34.55 | 34.9 |
GAD1-546 | 30.8 | 14.7 | 292.1 | 53.3 | 1.2 | 17 | 71.13 | 27.19 | 43.82 | 44.3 |
GAD1-594 | 52.2 | 31.6 | 351.7 | 66.1 | 1.2 | 16 | 50.53 | 15.69 | 52.76 | 53.3 |
FOXP2-647 | 28.3 | 27.7 | 311.2 | 54.0 | 1.1 | 17 | 82.47 | 14.62 | 46.68 | 47.2 |
Some of my best labeling results yet. The stock solutions are all almost twice as high as Simone's protocol calls for.
Hybridization[edit]
Sample Matrix[edit]
uL FOXP2-488 (pmol) | uL GAD1-546 (pmol) | uL GAD1-594 (pmol) | uL FOXP2-647 (pmol) | |
Sample A | 0 | 5 (220) | 0 | 5 (235) |
Sample B | 5 (175) | 0 | 5 (265) | 0 |
Protocol[edit]
- Prepare fresh paraformaldehyde 4% and keep at RT
- Fix sections with 4% paraformaldehyde for 15 minutes at RT
- Rinse sections twice with RT PBS
- Permeabilize sections in ethanol 70% EtOH; >90 minute incuabation at 4C
- Rinse sections twice with RT PBS
- Prepare the wash buffer and let it sit at RT
- Incubate the sections in wash buffer for 10 minutes at RT
- Warm hybridization buffer and warm it up to 37C
- Prepare hybridization reaction:
- hybridization buffer-100 uL; probe 2-10 uL
- Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.