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 22083
Data processing
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 CGS data extraction.
