characteristics.text: SAMN00165409
characteristics.characteristic: External Id
characteristics.text: LIBEST_022866
characteristics.characteristic: External Id
characteristics.text: Huazhong Agricultural University
characteristics.characteristic: INSDC center name
characteristics.text: 2008-03-21T00:00:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-10-11T17:45:57.607Z
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: early flowering mutant
characteristics.characteristic: cultivar
characteristics.text: The subtracted library was obtained by the juvenile wild type mRNAs as tester and cDNA from juvenile mutant mRNAs as driver. In January of 2005, the seeds of precocious trifoliate orange and wild type trifoliate orange were planted in 20 cm pots containing potting mix of a commercial medium and perlite at a ratio of 3:1. These juvenile trees were watered regularly with nutrient solution. Shoot meristem of the juvenile mutant and wild type trees were collected in March, June, September, and December, respectively. To prepare a representative sample of total RNA from the juvenile mutant and wild type in SSH process, different developmental stages of plant organs from roughly equal numbers of the juvenile mutant and wild type material were pooled, respectively. SSH library was performed using PCR Select cDNA Subtraction kit (Clontech, USA) according to the manufacturer's instructions. The final PCR products were purified using QIAquick PCR purification kit (Qiagen). The fragments obtained were cloned into the pGEM-T vector (Promega, USA) and used for the transformation of Escherichia coli DH5a. Individual clones from the library were randomly picked and stored in 384-well plates. A total of 3840 clones were collected in the forward subtracted and the reverse subtracted library. The inserts were amplified with the solution of 1ul single clone bacterial culture as PCR template, 0.5ul primer T7 (10umol/ul), 0.5ul primer SP6 (10umol/ul), 0.25 ul Taq DNA polymerase (Shanghai Sangon, China), 2.5 ul 10 x DNA polymerase buffer, 0.5 ul dNTPs (10mmo/ul) and 1.5 ul MgCl2 (25 mmol/ul), and added ddH2o to 25 ul. The PCR program is: 94 oC for 5 min; 94 oC for 30 s, 58 oC for 35s, 72 oC for 1.5 min, 30 cycles followed by a 5 min final extension at 72 oC.
characteristics.characteristic: description
characteristics.text: juvenile
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0000112
characteristics.characteristic: dev-stage
characteristics.text: Escherichia coli DH5alpha
characteristics.characteristic: lab-host
characteristics.text: Citrus trifoliata
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_37690
characteristics.characteristic: organism
characteristics.text: Shoot meristem
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PO_0006079
characteristics.characteristic: tissue-type
characteristics.text: juvenile early flowering trifoliate orange mutant subtracted cDNA library
characteristics.characteristic: title
characteristics.text: PGEM-T
characteristics.characteristic: vector