Daniel:Notebook/RNAFISH/2015-2-25
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Linnarsson Protocol (Started 2/24/2015)[edit]
Buffer Prep[edit]
- Hybridization Buffer
- RNAse free water 5.3 mL
- SSC 20X 1 mL
- Dextran sulfate 2 mL
- Formamide 1 mL
- E coli tRNA 500 uL
- RVC 200 mM (warm to 37) 100 uL
- BSA 50 mg/mL 40 uL
Ethanol Precipitation[edit]
- Centrifuge at 4 C for 30 minutes
- Remove supernatant and add 750 uL chilled 75% EtOH
- Centrifuge at minutes at 4 C for 10 minutes
- Dry the pellet in the hood; protect from light
- Resuspend pellet in 15 uL TE buffer
Dye Labeling Results[edit]
Column ng/uL | Column pmol/uL | Total Column DNA (ug) | Total Column dye (pmol) | Column dye/probe | Column Base:dye | EtOH Precip ng/uL | EtOH Precip pmol/uL | EtOH total DNA (ug) | EtOH total dye (pmol) | EtOH dye/probe | EtOH Base:dye | EtOH DNA Yield (%) | EtOH dye Yield (%) | Stock Concentration (uM) | |
FOXP2-488 | 24 | 6.8 | 2.4 | 680 | 1.9 | 11 | 90.6 | 11.4 | 1.4 | 171 | 0.8 | 24 | 56.63 | 25.15 | 11.4 |
GAD1-594 | 55.7 | 38.1 | 5.6 | 3810 | 4.5 | 4 | 96.9 | 14.1 | 1.5 | 211.5 | 1.0 | 21 | 26.10 | 5.55 | 14.1 |
So this is a great result, and also shows that there may be a considerable amount of unlabeled dye left after the spin column. For purposes of purification and increasing concentration, I should probably always do an Ethanol precipitation. It will also probably be necessary in the future to concentrate the probes even more, that is dissolve each probe in 5-7 uL and use it all in a go.
Hybridization[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 with ethanol (70% EtOH, >90 minute incuabation at 4C)
- Rinse sections twice with RT PBS
- Prepare the wash buffer and let it sit 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.