Total DNA from Xylella fastidiosa (isolate 187b), grown on solid PW medium
Biomaterial provider
Helvécio D. Coletta-Filho
Treatment protocol
Bacteria have been recovered from -70oC glycerol stocks and inoculated onto PW agar plates. Plates were maintained for 10 days at 28oC. The colonies were transferred once to new plates containing the same medium, grown for 20 days and harvested for DNA extraction
Growth protocol
Bacteria have been recovered from -70oC glycerol stocks and inoculated onto PW agar plates. Plates were maintained for 10 days at 28oC. The colonies were transferred once to new plates containing the same medium, grown for 20 days and harvested for DNA extraction
Extracted molecule
genomic DNA
Extraction protocol
Total genomic DNA was extracted according to : Dungan, D. (1989) in: Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K., Eds.), pp. 241-242. John Wiley and Sons, New York.
Label
Cy3-dCTP
Label protocol
DNA labelling was performed as described by: Behr, M.A., Wilson, M.A., Gill, W.P., Salamon, H., Schoolnik, G.K., Rane, S. and Small, P.M. (1999) Comparative genomics of BCG vaccines by whole-genome DNA Microarray. Science 284, 1520-1523.
Total DNA from Xylella fastidiosa (isolate 9a5c), grown on solid PW medium
Biomaterial provider
Helvécio D. Coletta-Filho
Treatment protocol
Bacteria have been recovered from -70oC glycerol stocks and inoculated onto PW agar plates. Plates were maintained for 10 days at 28oC. The colonies were transferred once to new plates containing the same medium, grown for 20 days and harvested for DNA extraction
Growth protocol
Bacteria have been recovered from -70oC glycerol stocks and inoculated onto PW agar plates. Plates were maintained for 10 days at 28oC. The colonies were transferred once to new plates containing the same medium, grown for 20 days and harvested for DNA extraction
Extracted molecule
genomic DNA
Extraction protocol
Total genomic DNA was extracted according to : Dungan, D. (1989) in: Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K., Eds.), pp. 241-242. John Wiley and Sons, New York.
Label
cy5-dCTP
Label protocol
DNA labelling was performed as described by: Behr, M.A., Wilson, M.A., Gill, W.P., Salamon, H., Schoolnik, G.K., Rane, S. and Small, P.M. (1999) Comparative genomics of BCG vaccines by whole-genome DNA Microarray. Science 284, 1520-1523.
Hybridization protocol
Labelled DNAs were mixed, dried in a Savant speed vacuum and resuspended in 100 ul of 1X Hybridization buffer, containing 6X SSC, 5X Denhardts solution, 0,25 mg/ml salmon sperm DNA, 0,01% SDS´and 50% formamide. Arrays were hybridized overnight (42oC) in a Gene-Tac Hybridization Station (Genomic Solutions, Inc., Ann Arbor, MI) and washed twice (42oC) in 0.5 X SSC, 0.01 % SDS, followed by two washes in 0.06 X SSC, 0.01 % SDS and two final washes in 0.06 X SSC. All washing steps consisted of 1 min of flow, followed by 5 min of incubation. Slides were then dried and subjected to fluorescent detection
Scan protocol
Slides were subjected to fluorescent detection with a GMS (Affymetrix) 418 Array Scanner (Affymetrix Inc., Santa Clara, CA) Cy3 channel was scanned at 100% laser power and 70% PMT gain Cy5 channel was scanned at 100% laser power and 80% PMT gain
Description
This hybridization is part of an experiment aimed at performing comparative genomic analyses among different isolates of Xylella fastidiosa
Data processing
Hybridized arrays were scanned in an Affymetrix 418 Array Scanner and images were analyzed with Affymetrix Jaguar v 2.0. Quality control of the hybridized spots was automatically performed by the software, based on spot morphology and local signal-to-background ratio, using the Easy Threshold and Variable Circle Size Algorithms. In all experiments, reliable hybridization signals could be obtained for more than 90% of the arrayed probes. Normalization between the intensities in the two channels was achieved with the Jaguar Control Spots option, using a list of 30 control ORFs that shared sequence identity in the genomes of strains 9a5c and Temecula-1.
