Cdna library construction kit

Total RNA of Zhongzhimian 2 cotton cultivar complete stool (grew under standard conditions) was extracted using a commercially available kit (Promega, Madison, WI, United States). Polyadenylated mRNA was separated with a PolyATract mRNA Isolation System (Promega, Madison, WI, United States). Subsequently, a cDNA library was generated by inserting fragments in a λZAP-II vector as the specifications of cDNA Library Construction Kit (Merck, Germany). The library was propagated on 140 mm plates to obtain about 10⁵ plaques (Wang et al., 2011a (link)). The conserved region of AtGLP3a isolated from Arabidopsis (Staiger et al., 1999 (link)), was labeled with ³²P-dUTP and used as probe for positive plaques by colony in situ hybridization. A positive plaque was obtained after three rounds, and the fragment was subcloned into pBlueScript II SK (+) through in vivo excision, following the protocol provided by manufacturer (Stratagene, United States).

Total RNA was extracted from C. komarovii complete stool using a commercially available kit (Promega, WI, USA). Polyadenylated mRNA was obtained using PolyATract mRNA Isolation System (Promega, WI, USA). Subsequently, a cDNA library was generated using a cDNA Library Construction Kit (Merck, Germany). The library was propagated on 150 mm plates to obtain about 10⁶ plaques [30 (link)]. A conserved 21-amino acid sequence (5’-FDXSYFHNKCLCGAPLPSCK-3’) at the C-terminus of all previously characterized PGIPs [31 ] was used to probe the library by colony in situ hybridization. A positive plaque was obtained after three rounds, and the fragment was subcloned into pBlueScript II SK (+) through in vivo excision, following the manufacturer’s protocol.