Methods And Materials Primer Design And Cloning - 768 Words

Methods and Materials: Primer Design and Cloning: To explore regions of the Drosophila genome that weren’t categorized by Stark lab fragments, we used a molecular approach to design novel candidate enhancers from the ChIP sequence peaks. This approach also allowed us to create narrower fragments (approximately 1000 bp), focusing on enhancer activity and reducing the chance of background activity. Fragments containing in vivo Bcd and Otd binding signals were selected by searching between 500 bp upstream to 500 bp downstream of the primary binding peaks from ChIP sequence data (Datta, Unpublished) for Bcd Early (BcdE), Otd Late (OtdL), Otd Early (OtdE), or both BcdE and OtdL. We designed primers for the top candidate enhancers using Primer3, calculating for appropriate GC content and melting temperature (primers ordered through Integrated DNA Technologies, Iowa). Restriction enzyme sites (AscI at 5` and BglII at 3’) were incorporated into the primers. Standard PCR amplification (Chen et al.) was performed with wildtype f ly genomic DNA and candidate primers. Qiagenâ„ ¢ kits were used for PCR purification and gel extraction. Vector Construction and Transgenesis: Reporter gene construction was conducted to obtain flies that contained the candidate enhancers. Reporter genes were constructed using the piB-HC-lacZ vector (Chen et al, 2012). The piB-HC-lacZ vector contains a 4.0 kb HindIII-SphI fragment, including an eve basal promotor, the lacZ-coding region, and the 3’ UTR from theShow MoreRelatedPolymerase Chain Reaction Essay1003 Words   |  5 PagesOne may view cloning as copying a living thing and producing multiple copies. People may think of cloning rabbits, sheep or humans. In the field of molecular biology, however cloning is viewed at a genetic molecular level, where a piece of DNA is copied on a large-scale by genetically copying tens to hundreds of thousands of identical DNA fragments. 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