characteristics.text: SAMN00164509
characteristics.characteristic: External Id
characteristics.text: LIBEST_022274
characteristics.characteristic: External Id
characteristics.text: Biotechnology Institute, Ankara University
characteristics.characteristic: INSDC center name
characteristics.text: 2007-12-04T00:00:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-10-11T17:45:40Z
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: Cabernet Sauvignon
characteristics.characteristic: cultivar
characteristics.text: Vitis vinifera roots grown in Murashige and Skoog (1962) macro and micronutrients with Gamborg's B5 vitamins (Gamborg et al., 1968) supplemented with 1.5% sucrose, 1% agar under control conditions as well as subjected to cold (0-1.5oC), water-deficit, and 150 mM NaCl stress for 2, 4, 6 h. cDNA Library was constructed using total RNA from cold, drought, 150 mM NaCl stressed and well watered Vitis vinifera roots. Total RNAs from different treatments were extracted and equal amounts were pooled before mRNA selection. Poly(A)+mRNA was isolated from total RNA using the Oligotex Direct mRNA kit (Qiagen). cDNA synthesis was conducted by coverting poly(A)+mRNA to double - stranded cDNA by using primer: 5'-AACTGGAAGAATTCGCGGCCGCTCGCATTTTTTTTTTTTTTTTTTV-3' (V=A,C,G) and Superscript III (Invitrogen). Double stranded cDNAs were size selected (more than 600 bp), adaptored with EcoRI adaptors (aattccgttgctgtcg - Promega # C1291) and digested with NotI. The cDNAs were then directionally cloned into EcoRI-NotI digested pBluescript II SK+ phagemid vector (Stratagene). The total number of white colony forming units (cfu) before amplification was 3x106. Blue colonies (empty vectors) were less than 10%. Purified plasmid DNA from the primary library was converted to single-stranded circles and used as a template for PCR amplification using the T7 and T3 priming sites flanking the cloned cDNA inserts as previously described (Bonaldo M, Lennon G, and Soares MB (1996) Normalization and subtraction: two approaches to facilitate gene discovery. Genome Research 9:791-806). The purified PCR products, representing the entire cloned cDNA population, were used as a driver for normalization. Hybridization between the single-stranded library (50ng) and the PCR products (500ng) was carried out for 44 hours at 30oC. Unhybridized single-stranded DNA circles were separated from hybridized DNA rendered partially double-stranded and electroporated into DH10B cells to generate the normalized library. The total number of clones with insert was: 1.6x106 cfu. Background of empty clones was less than 10%.
characteristics.characteristic: description
characteristics.text: 10 cm high plants grown in Magenta boxes
characteristics.characteristic: dev-stage
characteristics.text: Vitis vinifera
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_29760
characteristics.characteristic: organism
characteristics.text: EcoRI
characteristics.characteristic: re_1
characteristics.text: XhoI
characteristics.characteristic: re_2
characteristics.text: roots
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PO_0009005
characteristics.characteristic: tissue-type
characteristics.text: VVM
characteristics.characteristic: title
characteristics.text: Phagemid
characteristics.characteristic: v_type
characteristics.text: Lambda Uni-Zap XR, Bluescript SK-
characteristics.characteristic: vector