characteristics.text: LIBEST_009705
characteristics.characteristic: External Id
characteristics.text: SAMN00164211
characteristics.characteristic: External Id
characteristics.text: Washington University School of Medicine
characteristics.characteristic: INSDC center name
characteristics.text: 2001-08-16T00:00:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-10-11T17:39:44.703Z
characteristics.characteristic: INSDC last update
characteristics.text: live
characteristics.characteristic: INSDC status
characteristics.text: Generic
characteristics.characteristic: NCBI submission model
characteristics.text: Generic.1.0
characteristics.characteristic: NCBI submission package
characteristics.text: Bragg NTS382
characteristics.characteristic: cultivar
characteristics.text: The mRNA was isolated from roots of 8 day old 'Bragg' supernodulating mutant NTS382 seedlings that were infected with Bradyhizobium japonicum, strain USDA 110, 72 hours prior to harvest. Dr. Gary Stacey generously donated the tissue. The roots were flash-frozen in liquid nitrogen. Stratagene's cDNA Synthesis Kit (catalog number 200401) was used to synthesize the cDNA. First-strand synthesis was performed with 5-methyl dCTP, hence the ligated cDNA was hemimethylated. A modification of Stratagene's first-strand synthesis primer was used. An 'anchor' nucleotide (V=A, C, or G) was added to the 3' end of the primer [GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAG(T)18V] to anchor the primer at the 5' end of the poly(A) tract. After second-strand synthesis, the cDNA ends were filled in with cloned Pfu DNA, ligated to EcoRI adapters and subsequently phosphorylated. The cDNA was then precipitated and redissolved in sterile, RNase-, DNase-free water. The XhoI site within the first- strand synthesis primer was then restricted by digestion with XhoI from Promega (40U/ul); all XhoI sites in the cDNA would be protected by their hemimethylated status. The cDNA constructs were size-fractionated with a 500bp cutoff, using Sephacryl S-500 High Resolution (Pharmacia Biotech) in a 2-mm diameter column and a bed volume of approximately 1ml. The column eluent was precipitated, redissolved, and ligated into Stratagene's pBluescript II XR Predigested vector (pBluescript II SK(+) vector that has been digested with EcoRI and XhoI, and phosphorylated by Stratagene). This library was constucted in the laboratory of Dr. Paul Keim and Dr. Virginia H. Coryell at Northern Arizona University.
characteristics.characteristic: description
characteristics.text: 8 days old
characteristics.characteristic: dev-stage
characteristics.text: DH10B
characteristics.characteristic: lab-host
characteristics.text: Glycine max
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_3847
characteristics.characteristic: organism
characteristics.text: EcoRI
characteristics.characteristic: re_1
characteristics.text: XhoI
characteristics.characteristic: re_2
characteristics.text: Roots of 8 day old 'Bragg' supernodulating mutant NTS382 seedlings
characteristics.characteristic: tissue-type
characteristics.text: Gm-c1080
characteristics.characteristic: title
characteristics.text: plasmid
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/SO_0000155
characteristics.characteristic: v_type
characteristics.text: pBluescript II SK+
characteristics.characteristic: vector