RESOURCES

Resources developed during the project will be available to the research community on request. These are:-

Flounder Gene Libraries:
cDNA Libraries:

Tissue Vector Titre Details
Liver pTriplEx2   Immature fish, Normalised*
Intestinal mucosa pTriplEx2   Immature fish, Normalised*
Whole kidney (head+ trunk) pTriplEx2   Immature fish, Normalised*
Induced Liver pTriplEx2   Immature, Induced§, Normalised*

*Construction of the “Normalised” cDNA libraries was achieved by combining the protocols of Carninci (1999 ) together with the efficiency of a SMART™ library construction kit (Clontech, UK) in generating full length cDNA libraries.
Tissue mRNA was purified and one sample was biotinylated to produce “Driver” DNA, first strand cDNA was synthesised from another aliquot using a Clontech SMART™ cDNA synthesis kit to genrate full length “tester” cDNAs. The two were hybridised at 42°C at Rot of 250 so that the abundant classes of mRNA reassociated more rapidly than the lower abundance mRNA species (2nd order kinetics). The cDNA-biotin labelled-mRNA complex was then removed with streptavidin-coated magnetic beads to leave a normalised cDNA sample in solution. This was then used for second strand synthesis, digested with SfiI and was ligated into lambdaTriplEx2 (Clontech) and recombinant lambdaTriplEx2 was converted to pTriplEx2


§ “Induced” libraries were produced by pooling RNA samples extracted from fish tissues 48h post-treatment with 3-MC, Arochlor 1254, E2, 3-methyltestosterone, PFOA, Cd , PCN and tBHP (doses as per pollutant page, 3 fish per time/treatment)

Genomic DNA Library:

Lambda FixII (Lee & Chipman, 1997)

Microsatallite libraries:

Flounder GGAT/GATA/GACA repeat enriched, pBluescript KS+, NotI linkers.

Flounder TAA/AGC repeat enriched, pBluescriptKS+, NotI linkers.

SSH clones:

Flounder from Benzo(a)pyrene, cadmium and BaP+ Cd treatments.