Pgem t vector cloning kit

Outward facing primers were used against exons 2, 4, 6, 7, 8, 14 and 16 with forward primer against the 3′ end of each exon and reverse primer against 5′ end of the same exons. RT-PCR conditions used were the same as mentioned before. When multiple bands were detected, cloning of the PCR products was conducted using a pGEM^®-T Vector cloning kit (Promega, Madison, WI, USA). Sequencing of the resulting clones using T7 and SP6 primers was performed in two directions using ABI Prism 3730xl Genetic Analyzer as above.

The gel slices containing PCR amplicons were purified using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, USA). The eluted products were cloned into pGEM-T vector cloning kit (Promega) and transformed into the top ten Escherichia coli cells. Blue-white screening was observed by adding X-gal (100 mg/µl) and Isopropyl β- d-1-thiogalactopyranoside (50 mg/µl) on Luria Broth plate. Recombinant clones were confirmed by colony PCR using gene-specific primers. The plasmid DNA was extracted using the plasmid DNA extraction kit (Thermo Fisher Scientific, USA) and sequenced (Eurofins, Bengaluru). The forward and reverse sequences were edited and submitted to GenBank. Sequences obtained from the current study and other GenBank sequences were aligned by ClustalW using the neighbor-joining method [20 (link)] and the phylogenetic tree was constructed using MEGA 10.0.5 [21 (link)].