Cells were cultured in Todd-Hewitt broth (THB)(Difco Laboratories, Detroit, MI) medium
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted using QIAGEN Genomic-tip 100/G kit (Qiagen)
Label
Cy3
Label protocol
Genomic DNA was labeled with a Random prime reaction. DNA (1 ug) was mixed with 1 O.D. of 5’-fluorescence dye labeled random nonamer (Cy3 for test strains and Cy5 for reference strain) (TriLink Biotechnologies) in 62.5 mM Tris-HCl, 6.25 mM MgCl2 and 0.0875% ß-mercaptoethanol, denatured at 98°C for 5 min, chilled on ice, and incubated with 100 units Klenow fragment (NEB) and dNTP mix (6 mM each in TE) for 2 h at 37°C. Reactions were terminated with 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water. A fifty-fold amplification was typically achieved.(http://www.nimblegen.com/products/lit/lit.html)
Cells were cultured in Todd-Hewitt broth (THB)(Difco Laboratories, Detroit, MI) medium
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted using QIAGEN Genomic-tip 100/G kit (Qiagen)
Label
Cy5
Label protocol
Genomic DNA was labeled with a Random prime reaction. DNA (1 ug) was mixed with 1 O.D. of 5’-fluorescence dye labeled random nonamer (Cy3 for test strains and Cy5 for reference strain) (TriLink Biotechnologies) in 62.5 mM Tris-HCl, 6.25 mM MgCl2 and 0.0875% ß-mercaptoethanol, denatured at 98°C for 5 min, chilled on ice, and incubated with 100 units Klenow fragment (NEB) and dNTP mix (6 mM each in TE) for 2 h at 37°C. Reactions were terminated with 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water. A fifty-fold amplification was typically achieved.(http://www.nimblegen.com/products/lit/lit.html)
Hybridization protocol
Labeled genomic DNA was hybridized to arrays in the NimbleGen Hybridization Buffer for 16 hours at 42 ℃ using a MAUI hybridization system (BioMicro Systems, Inc. Salt Lake City, Utah). Labeled genomic DNA (5ug) from the reference strain Streptococcus suis 89/1591 and from each test strain were co-hybridized to each array. Arrays were washed with NimbleGen wash buffer, and then were spun dry in a microarray high-speed centrifuge (TeleChem International, Inc., Sunnyvale, CA) and stored until scanned.(http://www.nimblegen.com/products/lit/lit.html)
Scan protocol
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol
Description
Mutation mapping 89-2479
Data processing
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 CGS data extraction.
