title: Mesorhizobium array [MrNa1]
status: Public on May 08 2015
contact.city: Ottawa
contact.first: Linda
contact.middle: J
contact.last: Harris
contact.email: Linda.Harris@agr.gc.ca
contact.state: Ontario
contact.address: 960 Carling Ave.
contact.department: Eastern Cereal & Oilseed Research Centre
contact.country: Canada
contact.institute: Agriculture & Agri-Food Canada (AAFC)
contact.phone: 613-759-1314
organism: Mesorhizobium sp. N33
description: Spotted PCR amplified from cloned genomic DNA digests. Each array has 48 blocks. Each block has 17 rows and 16 columns. Arrays were printed using the BioRobotics MicroGrid Compact spotter using Microsoft 10K split pins. Features vary from 70-110μm in diameter.
  
  Please note that only a selection of clones have been sequenced and their sequences are provided in the 'platform_sequences.txt'.  The sequences are not of the entire clone (from one or both ends using SL1/SR2 primer). The sequences were assembled together to form contigs and to discover which clones were overlapping genomic sequences.
manufacture_protocol: Arctic strain N33 cells were grown in 100 ml of YMB media with constant shaking (150 rpm) at room temperature (~21°C) to mid-log phase (OD600= 0.4-0.6) and cells were precipitated and washed in TES buffer (Gray et al., 1990) to eliminate extra-cellular polysaccharides ,by centrifugation (10,000g) at 4°C. Whole genomic DNA isolation was performed as described by Laberge et al., (1989) and stored at -20°C. DNA quality was verified using a NanoDrop 1000 spectrophotometer and by agarose gel (1%) electrophoresis. Two μg of DNA was mechanically sheared (Burger et al., 2007) using a nebulizer (Unomedical INC, McAllen, Texas USA, Ref #950-E) under nitrogen pressure of 20 psi for 3 minutes. DNA fragments ranging from 0.3-2 kb were subsequently blunt-ended using T4 DNA polymerase and Klenow DNA polymerase (Lucigen, Mississauga, Ontario). Blunt-ended DNA fragments (300 ng) were ligated to 50 ng/μl of blunt-ended pSMART-HC Kan vector (Lucigen,) and transformed into electro-competent E. coli DH5α cells (BIO-RAD). Plasmid inserts were amplified by standard PCR from 5760 clones (Biometra thermocycler) using the universal forward (SL1=5'-CAGTCCAGTTACGCTGGAGTC-3') and reverse primers (SR2=5'-GGTCAGGTATGATTTAAATGGTCAGT-3') complementary to vector sequences flanking the DNA inserts. Clones were digested with EcoRI (Boehringer Mannheim, Laval, Quebec, Canada) and fragment size and purity were evaluated by agarose gel electrophoresis. Some inserts were sequenced (Sanger et al., 1977) to verify that they were bona fide Rhizobium sequences by homology with the database GenBank. All recombinant E. coli clones were stored in glycerol at -85°C. Recombinant plasmid and PCR amplicons were stored at -20°C. PCR reactions from the Mesorhizobium strain N33 library were purified using the QIAquick 96 PCR Purification Kit (Qiagen, Cat. #28183) and transferred into the 384 master plate format using a Multiprobe II EX Robotic Liquid Handling System (Perkin Elmer, Shelton, CT). Plates were then dried without heat in a DNA 120 Speed Vac (Savant Instruments Inc., Holbrook, NY). Dried samples were resuspended in 10μl of 3XSSC and stored at -80°C. A total of 13,056 features, consisting of duplicate spots for each clone and 32 spike-in and negative controls for each block were arranged and printed on Corning GAPS II coated slides (Cat. # 40003), as per Corning instructions (www.corning.com/lifesciences). Arrays were printed using the BioRobotics MicroGrid Compact spotter (Digilab Inc, Holliston, MA). The arrays were snap-dried and DNA UV cross-linked to the slides using a CL-1000 ultraviolet cross linker (UPV, Upland, CA) for a total of 300 mJ.