| Products/Services Used | Details | Operation |
|---|---|---|
| Peptide Synthesis | For this purpose, a synthetic sequence was ordered from GenScript (http://www.genscript.com), comprising an attB1 sequence, followed by the SP1–2 signal peptide sequence (Futatsumori-Sugai and Tsumoto, 2010), a BamHI site, a c-myc-tag, a codonoptimized GST-sequence, and an attB2 sequence. The fragment was amplified by PCR (PHusion DNA Polymerase from Thermo Scientific, Primers were ordered at Microsynth) and subsequently gel purified (For Agarose gel electrophoresis, Gene Ruler Mix DNA ladder from Thermo Scientific as well as the 1kb DNA ladder from Biolabs were used). In a total volume of 8 µl, 150 ng of the PCR product were mixed with 180 ng of purified pDONR221 DNA (Life technologies) and 4.5 μl TE buffer pH 8.0. Then, 2 µl BP clonase II enzyme mix (life technologies) was added and the recombination step was carried out according to the standard Gateway protocols. The BP recombination reaction facilitates transfer of a gene of interest-in this case the attB_Signal_C-myc_GST PCR product-to an attP containing vector as pDONR221. Finally, DH5α Chemically Competent E. coli were transformed with the BP reaction mix and recombinant progeny was selected on LB containing 50 μg/ml kanamycin. To enable selection of entry clones, the pDONR221 vector on the one hand contains a Kanamycin resistance gene, on the other hand the two att sites are flanking a cassette containing the ccdB gene for negative selection. | Get A Quote |
Fibropapillomatosis (FP) is a neoplastic disease and a most important cause for stranding and death of the green turtle chelonia mydas. The Chelonid Herpesvirus 5 (ChHV5) is considered its causative agent. Since ChHV5 does not replicate in cell cultures, many questions concerning its pathogenesis and epidemiology, including the prevalence among marine turtles have not been solved. I developed an ELISA for detecting turtle antibodies against two putative ChHV5 envelope glycoproteins (F-US4 and F-US8). To generate standard antisera for cut-off values, I produced soluble fragments of these antigens with the aim of using them as immunizing antigens in hatchling turtles. To ask from which age on hatchlings would be ... More
