ASPA Gene PCR

RNA extracted from zebrafish embryos at 24, 48 and 72 hours post fertilization (hpf); 5 and 10 days post fertilization (dpf) was used to synthesize cDNA. Primers designed to amplify 671bp sequence of ASPA gene utilized to run PCR and determine temporal expression patterns. Results visualized using gel electrophoresis and PCR cleanup performed to send sample for sequencing.  

Gene Cloning

ASPA gene from PCR was cloned into a pDRIVE plasmid by a ligation reaction. Transformation of E. coli with pDRIVE plasmid performed; kanamycin resistance screening used to confirm transformation success. Plasmid DNA isolated using Miniprep and sent for sequencing to confirm presence of ASPA gene in plasmid. PCR conducted using M13 primers and product purified for riboprobe synthesis.