Most methods for isolation of RNA from yeast require tedious vortexing with glass beads, and give low yields when scaled down to 10 ml cultures (1). In addition, it is frequently desirable to prepare RNA from several different yeast strains grown under a variety of growth conditions, and preparations using glass beads are impractical when dealing with multiple samples.Heating and freezing of yeast cells in the presence of phenol and SDS has been used for large scale isolation of RNA from yeast (2). We report here a mini-prep version of yeast RNA isolation using phenol and SDS that allows for the processing of a dozen samples in about 60 minutes, and provides enough RNA to run several northern blots. In our hands methods using glass beads are slower and give lower yields of RNA.RNA was isolated as follows. Ten ml cultures were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD6W of 2.5-5.0. We have also used YP 2% galactose, YP 4% ethanol/3 % glycerol, YP 2% raffinose and a variety of minimal media. The cells were harvested by centrifugation and resuspended in 400 td of 50 mM Na acetate pH 5.3, 10 mM EDTA ('AE buffer').The resuspended cells were transferred to a 1.5 ml microcentrifuge tube and 40 IAI of 10% SDS was added. The suspension was vortexed and an equal volume of fresh phenol, previously equilibrated with AE buffer, was added. The mixture was again vortexed and incubated at 65°C for 4 min. The mixture was then rapidly chilled in a dry ice/ethanol bath until phenol crystals appeared, and then centrifuged for 2 min at maximum speed in a microcentrifuge to separate the aqueous and phenol phases. The upper, aqueous phase was transferred to a fresh microcentrifuge tube and extracted with phenol/chloroform at room temperature for 5 min. The extracted aqueous phase was then brought to 0.3 M Na acetate, pH 5.3, by adding 40 tl of 3 M Na acetate pH 5.3, after which 2.5 volumes of ethanol were added to precipitate the RNA. After washing with 80% ethanol, the pellet was dried and resuspended in 20 i1 of sterile water and stored at -70°C until used. Throughout the preparation, normal precautions to avoid ribonuclease contamination were taken (3).The isolation procedure was performed using seven different wild type yeast strains and over two dozen derivatives of these strains. Yields varied between 60 ,ag and 300 Mg of RNA per 10 ml culture, with an average yield of 135 ,ug as quantitated by absorbance at 260 nm. Fig. la shows the quality of the RNA that is isolated using this method. No degradation of ribisomal RNA bands is seen, and recoveries of both high and low molecular weight RNA appear excellent.

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the ‘Janelia Fluor’ (JF) series of dyes. Here, we refine and extend this strategy, showing that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties with unprecedented precision. This strategy yields a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red with utility in cells, tissue, and animals.

Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.