characteristics.text: LIBEST_003798
characteristics.characteristic: External Id
characteristics.text: SAMN00158351
characteristics.characteristic: External Id
characteristics.text: Washington University School of Medicine
characteristics.characteristic: INSDC center name
characteristics.text: 2000-03-13T00:00:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-10-11T17:36:42.530Z
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: Williams
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0007023
characteristics.characteristic: cultivar
characteristics.text: This cDNA library was constructed from mRNA isolated from cotyledons of 8-day-old 'Williams' seedlings which were propagated on paper towels with distilled water for 3 days (etiolated), then greenhouse grown for 5 days in potting soil. The cotyledons 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 500 bp cutoff, using GibcoBRL Life Technologies' cDNA Size Fractionation column. The column eluent was then 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). 100% of the white and 87.5% of the blue colonies appear to contain recombinant plasmids with cDNA inserts, based on size (n=28 and 8 respectively). This library was constructed by Dr. Paul Keim and Dr. Virginia Coryell.
characteristics.characteristic: description
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: Cotyledons of 8-day-old 'Williams' seedlings
characteristics.characteristic: tissue-type
characteristics.text: Gm-c1032
characteristics.characteristic: title
characteristics.text: plasmid
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/SO_0000155
characteristics.characteristic: v_type
characteristics.text: pBluescript II XR
characteristics.characteristic: vector