Data normalilsed within the statistical programming environment R using the package LIMMA. Arrays were normalised using the within-array global median function followed by the across-array scale function
Agilent Technologies Image Analysis and Feature Extraction software (Version 10.5)
For hybridisation on 105k arrays, 30-40 pmols of cDNA-sample was mixed against 30-40 pmols of wt gDNA-sample. The volume was adjusted to 260 uL with sterile nuclease-free water and 26 uL of blocking agent was added in each tube and the samples were vorte
use of an Agilent dual laser 5 um scanner; at 532 nm for the cy3 channel and 635 nm for the cy5 channel.
Fluorescently labelled cDNA was produced for each RNA sample using reverse transcription; 10 ug of RNA was used for each cDNA synthesis/ labelling in 30ul total volume with 5.1 ug of random primers (Invitrogen). The RNA-primer mix was denatured at 70 degrees C fo
Prior to RNA extraction the mycelium was treated with RNA Protect Bacterial Reagent (Qiagen); 20 ml culture supernatants were removed and 4 ml RNA Protect was added, samples were agitated on a vortex mixer for 5 s, left at room temperature for 5 min and p
Incubate at 30 degrees C in 500 ml baffled flasks.
Fluorescently labelled genomic DNA was generated using 2-3 ug gDNA (S. coelicolor M145) that was added to 3 ug random primers in a total volume of 41.5 ul, and denatured at 95 degrees C for 5 minutes. 1ul of dNTP mixture 5 mM dATP, dGTP, dTTP, 2mM dCTP, 1.5 ml C
The procedure followed was essentially as described in Kieser et al. (2000). Streptomyces cultures were grown for 36-40h in 25 ml YEME at 30 degrees C. Cultures were centrifuged (2000 ×g, 10 min) and the supernatant discarded, remaining pellets were washed in 15
Add 50-100 ul spore suspension into 50 ml YEME plus 10% sucrose and MgCl2 and glycine supplement as specified in Kieser et al., 2000. Incubate in a shaking incubator at 30 degrees C for 36-42 h in 250 ml flasks with springs up to early stationary phase (OD450 ~ 2
Strains were grown on sporulating MS agar medium (Hobbs et al., 1989) for 5-7 days at 30 degrees C. Streptomyces spore suspensions were prepared as described by Kieser (2000) et al., for future inoculation.
Add 4.00E+06 spores to 160 ml of Nurtient Broth (NB).