Construction of impt-1 RNAi Plasmid

Construction of impt-1 RNAi plasmid was similar to that reported elsewhere [55 (link), 56 (link)]. A region corresponding to an exonic part of the impt-1 gene of N2 worms was amplified by PCR using the forward primer 5′-AATCTAGACTGCCGGGCAACACATAAT-3′ and the reverse primer 5′-AACTCGAGTATCCGCTTTCATTCGATCC-3′. The resulting PCR product was cloned into the L4440 vector using the XbaI and XhoI sites (regions in bold) and the Quick Ligation kit (New England Biolabs). Plasmids were transformed into E. coli TOP10 (One Shot® iTOP10 Chemically Competent E. coli). Recombinant clones were selected using ampicillin. Plasmids were extracted using QIAGEN Plasmid Mini Kit and sequenced to confirm the presence of the insert. Plasmids were used to transform E. coli HT115(DE3), which was used to feed the nematodes.