biomaterial: SJY6 yeast strain, exponential growth in YPD.
Growth protocol
Exponential growth in YPD.
Extracted molecule
total RNA
Extraction protocol
Total RNA isolated from yeast cells was prepared as described by Sherman et al. [1986], but using a multiple-sample automated device (Fast-Prep, BIO101) to break the cells. Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Spring Harbor Cold Laboratory Press, Cold Spring Harbor, New York
Label
33P
Label protocol
About 30-40 µg of total RNA were reverse transcribed into cDNA by adding 200 us of RT polymerase SuperScript II and random hexamers (Invitrogen), 1 µL RNaseOUT (Invitrogen), 6 µL 5X First Strand Buffer (Invitrogen), 1.5 µL dNTP mix (16 mM dATP, dTTP, dGTP, and 100 µM dCTP) and 5 µL [α-33P]dCTP (3000 Ci/mmol, 10 µCi/microL) in a final reaction volume of 30 µL. The labeling reaction was incubated for 1 h at 43ºC. 1 µL of EDTA 0.5 M was added to stop the reaction. The labeled cDNAs were purified by a S300-HR MicroSpin column (Amersham BioSciences).
Hybridization protocol
Hybridization Solution was: 5X SSC, 5X Denhardt’s, 0.5% SDS and 100 µg herring sperm DNA/ml. The hybridization protocol used was as follows. Filters were inserted in 12.5X 2.5-cm flat-bottom plastic tubes and pre-hybridized in a rotator oven with 5 ml pre-hybridization solution (the same as used for hybridization but without the radioactive sample) at 65ºC. The pre-hybridization solution was then replaced with 5 ml of the same solution containing 3X10exp6 dpm/mL of radioactive sample and hybridized for 44h. Washing conditions were: 1h at 65ºC for 20 min in 2X SSC, 0.1% SDS, and twice at 65ºC for 30 min in 0.2X SSC, 0.1% SDS.
Scan protocol
Images were acquired using a FujiFilm FLA3000 Phosphorimager. After the washing step, membranes were kept humid, sealed in Saran wrap, avoiding any bubbles, and exposed to an imaging plate (BAS-MP, FujiFilm) for various times. Filters were stripped by pouring 3X150 ml boiling stripping buffer (5 mM sodium phosphate, pH 7.5, 0.1% SDS) over the membrane. The first time, the stripping buffer was immediately changed while after the second and third washes the filters were left to cool at room temperature. To ensure that radioactivity had been eliminated, the filters were either checked with a Geiger counter or re-scanned with the Phosphorimager.
Description
Total RNA from SJY6 yeast strain. Control time before adding doxycycline. Hybridization on filter 16. Replicate 3.
