title: S. uberis-specific array
status: Public on Dec 31 2016
contact.city: Bundoora
contact.first: Nitin
contact.last: Mantri
contact.email: nitin.mantri@rmit.edu.au
contact.state: Victoria
contact.address: Plenty Road
contact.department: School of Science
contact.country: Australia
contact.institute: RMIT University
organism: Streptococcus uberis
manufacture_protocol: Streptococcus uberis isolates from cows with clinical mastitis and belonging to MLST types associated with virulence were selected as the source of genomic tester DNA. Isolates from cows with low cell counts and thought to represent possible non-virulent isolates were selected as the source of genomic driver DNA for suppression subtractive hybridization. All the isolates were collected during a previous study conducted at RMIT laboratory by Tomita et al. (2008). The tester pool consisted of six isolates belonging to GCC ST5 and GCC ST143, while the driver pool consisted of six isolates belonging to GCC ST86s.   The gDNA subtraction, library and microarray construction were performed according to the procedure described by Jayasinghe et al. (2007). Subtraction was conducted on the basis of manufacturer’s instructions (Clontech, 2008) described in the PCR-Select™ cDNA Subtraction Kit. Equal amounts of DNA extracted from six clinical mastitis isolates represented the tester pool while the driver pool was represented by equal amounts of DNA extracted from six low cell counts isolates.Subsequently, 4 μg of pooled gDNA from each group was restriction digested in a 50 μL reaction using 5U of Rsa I. As previously described (Jayasinghe et al., 2007), the digested driver gDNA pool was subtracted from digested tester gDNA pool to isolate tester-specific DNA using the Clontech PCR-SelectTM cDNA Subtraction Kit (Clontech, Mountain View, CA).The tester-specific DNA fragments were cloned into pGEM-T® Easy Vector (Promega, Madison, WI) and transformed into Escherichia coli JM109 competent cells (Promega, Madison, WI), resulting into 280 clones with insert of 250-750 bp.                                              Tester-specific clone amplification and SDA printing  Following the PCR amplification, approximately 80 µL of PCR products were transferred to V-bottom polypropylene 96-well plates. Using a multichannel pipette 96% of ethanol was added to a final concentration of 59% and 3M sodium acetate (pH 5.2) to a final concentration of 90 mM and the plasmid DNA was precipitated overnight at – 20oC. The plates were then centrifuged at 3000 rpm for 1.5 hr at 4oC. The supernatant was decanted using a pipette and the plates were inverted briefly to drain. The plasmid DNA pellet was washed with 70% ethanol prior of centrifugation and draining with the same conditions, the pellets air dried at room temperature for ≈2 hr. The plasmid DNAs were then resuspended in 50% dimethyl sulfoxide (DMSO) and stored at 4oC until use.   A total of 280 clones with inserts ranging from 250 bp to 750 bp, were transferred into a 384-well plate including the positive and negative controls. The control spots on the SDA included printing control (Cy-3) and negative controls; printing buffer (50% DMSO), nested PCR primers 1 and 2R, and pGEM®-T Easy vector. The positive controls consisted of three housekeeping genes; hasA, hasC, and pauA. The 280 clones together with the controls were printed on aminosilane-coated slides using a BioRobotics® MicroGrid II Compact arrayer (Genomics Solutions, Ann Arbor, MI) microarray spotter at RMIT University, Australia.