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Ovary Library

Library: cLED
TIGR ID: TOV
Authors: Alcala, Vrebalov, White, and Giovannoni
Date made: 11/98
Species: Lycopersicon esculentum
Accession: TA496
Tissue: carpel
Developmental stage: 5 days pre-anthesis to 5 days post-anthesis
Vector: pBluescript SK(+/-)
Host: SOLR
Primary pfu: 1.0 x 106
Number mass excised: 3.0 x 105
Average insert length: 1.5 Kb
Cloning sites: 5' EcoRI, 3' XhoI
Antibiotic: ampicillin
Primers: M13F and M13R
Comments: Construction of an early carpel (pre-anthesis through 5 days post-anthesis) cDNA library has been completed. Approximately one millions unamplified clones were generated and 300,000 were used in mass excision. Forty 384-well plates (15,360 clones) were picked. On hundred random clones were miniprepped and PCR'd with an average insert size of 1.5 Kb (range 0.8-2.5 Kb). XhoI and EcoRI digests of the same 100 clones were performed. Only two clones were identified with internal XhoI and EcoRI sites, which may be indicative of chimeric ligation, though double digestion suggested they were not chimeras (the internal sites are both at least several hundred bp internal, but not adjacent). 48 of the clones were sequenced.Flowers were collected from about 25 TA496 plants every two days for one month. Flowers were approximately 5 days pre-anthesis (based on tagging) through 5 days post-anthesis (based on tagging and appearance). At 5 dpa, the carpels were about 0.5 cm in diameter. Flowers were collected at five different stages: 1)5 days pre-anthesis, 2) anthesis (based on partial sepal separation and anther tube swelling, 3) 5 dpa, 4) intermediate between 1 and 2, and 5) intermediate between 2 and 3. About 0.5 grams of tissue was collected from hundreds of flowers in the early stages, while about 10 grams of tissue was collected from about 10 stage-5 carpels. 0.5 to 1 gram per stage was combined and used for RNA isolation. The carpels included ovules and embryos. The anthers from the first three stages and some of the fourth stage were collected for an anther library.