Extraction of RNA，Dot Blots，and Northern Hybridiza...
R.sphaeroides RNA was isolated and extracted from cells as described previously［10］.Dot blots and Northern hybridizations with nick-translated probes were performed according to the previously described procedures［14］.Dot blots were used for quantitative assessment of cellular mRNA levels in response to light and O2.The Northern hybridization provided additional information on the number，sizes，and the relative amount of the individual puf transcripts.Quantitation was performed by scintillation counting of the filters or densitometer scans of autoradiograms well within the linear response of the X-ray film.The levels of specific cellular mRNA species determined by dot blots and Northern hybridizations were in agreement.
Northern hybridizations with 32P-labeled deoxyoligonucleotide probes were performed as follows.The hybridization buffer was essentially that described by Wallace et al.［15］and consisted of 90 mmol/L Tris-Cl（pH 7.5），0.9 mol/L NaCl，6 mmol/L EDTA，0.5%%sodium dodecyl sulfate，10%dextran sulfate，5×Denhardt's，100 μg/ml Escherichia coli tRNA，and 1 mmol/L ATP to minimize background hybridization from unincorporated［γ-32 P］ATP used to label the deoxyoligonucleotide probes.The preheated（55C）hybridization buffer was passed through a 0.45 μm nitrocellulose filter prior to use to reduce background hybridization.GeneScreen filters were prepared for hybridization by a 1-h incubation with hybridization buffer at 55℃followed by at least a 1-h incubation at room temperature（23℃）prior to the addition of the labeled deoxyoligonucleotide probes.Hybridization was conducted at room temperature for 12—16 h followed by washing of the filter（2 washes of 5 min each）at the indicated temperature in 6×SSC.While still moist，the washed filters were covered with plastic wrap for exposure to X-ray film；the use of moist filters facilitates the removal of nonspecifically bound probes in subsequent higher stringency washes at elevated temperatures.(https://www.daowen.com)
