Resources developed during the project will be available to the
research community on request. These are:-
Flounder Gene Libraries:
kidney (head+ trunk)
*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)
Lambda FixII (Lee &
repeat enriched, pBluescript KS+, NotI linkers.
Flounder TAA/AGC repeat
enriched, pBluescriptKS+, NotI linkers.
Flounder from Benzo(a)pyrene,
cadmium and BaP+ Cd treatments.