title: Asterids-specific array
status: Public on Jul 31 2014
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: asterids
description: specific for all species belonging to Asterids clade of plants
manufacture_protocol: Plant material and genomic DNA extraction : Leaf tissues of 67 species representing the Asterid clade (AC) and 104 species representing the non-asterid angiosperms and non-angiosperms (NA) were obtained from the herbarium at Southern Cross University Plant Science, NSW, Australia. High quality genomic DNA (gDNA) was extracted from these specimens using DNeasy® Plant Mini Kit (Qiagen Inc., Australia). The quantity of DNA was measured using Eppendorf spectrophotometer whilst the quality/integrity was assessed by 1.5% agarose gel electrophoresis.
  Genomic DNA subtraction and library construction : The gDNA was pooled into two separate groups, AC and NA, by mixing equal quantities of DNA from individual species belonging to that group. Subsequently, 4 μg of pooled gDNA from each group was restriction digested in a 50 μL reaction using 5U of HaeIII and AluI (New England Biolabs). As previously described (Jayasinghe et al., 2007), the digested NA gDNA pool was subtracted from digested AC gDNA pool to isolate AC-specific DNA using the Clontech PCR-SelectTM cDNA Subtraction Kit (Clontech, Mountain View, CA).  The AC-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 283 clones with insert of 250-750 bp.
  AC-specific clone amplification and SDA printing : The 283 AC-specific DNA clones were amplified in 100 μL PCR reactions using Clontech nested primers as described (Jayasinghe et al., 2007). PCR products were transferred into V-bottom polypropylene 96-well plates and purified by ethanol/sodium acetate precipitation before resuspending in 50% dimethyl sulfoxide (DMSO). The control spots on the SDA included printing control (Cy-3) and negative controls viz., printing buffer (50% DMSO), nested primer 1 and 2R (Clontech, Mountain View, CA), and pGEM-T® Easy Vector (Promega, Madison, WI) digested with HaeIII and AluI. The 283 AC-specific DNA fragments along with controls were spotted on a Corning GAPS II coated slides (Corning Incorporated Life Sciences, Acton, MA) using BioRobotics MicroGrid II Compact (Genomics Solutions, Ann Arbor, MI) microarray spotter at RMIT University, Australia.