Shelly’s Notebook: Wed. Oct 31, 2018

-Geoduck hemolymph extraction

-Met with Jose – he has experience with developing Vitellogenin ELISA assays in fish – Options: – ELISA: quantitative – Western Blot: not quantitative – qPCR: quantitative – Calcium assay; calcium correlated with vitellogenin (vitellogenin binds Ca2+) – assay for Upstream vitellogenesis inducer? (i.e. estradiol, although Brent says Geoducks likely don’t have this) – To develop ELISA: – need 1) isolated vitellogenin and 2) antibody. Need isolated protein to generate standard curve and generate antibody – can isolate vitellogenin by: – fractionation? It’s large – inducing expression – cloning and doing bacterial expression – can generate antibody by sending protein (or protein fragment) to commercial antibody lab (they inject rabbits) – To develop Western: – only need antibody – Calcium assay

 - To develop qPCR: - need primers - need sequence - VTG superfamily domain? -BLASTX to get DNA sequence? -revtrans on serial cloer to get DNA sequence? - BLAST to Geoduck genome - build BLAST index out of Geoduck genome - align VTG superfamily domain to genome - align protein to genome - samples: - no template - non-DNase treated sample or gDNA prep? - RNApreps - hemocytes from different stages - immature - early - middle - late - post spawn - gonad tissue from different stages - immature - early - middle - late - post spawn - hepatopancreas - immature - early - middle - late - post spawn  

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