Pgadt7 rec plasmid

Error-prone PCR mix was composed of G2-polymerase buffer (Promega) and G2-polymerase, 5 mM MgCl₂, 0.15 mM MnCl₂, unbalanced dNTP mix (2.5 mM dATP, 2.5 mM dGTP, 10 mM dCTP, 10 mM dTTP). The pool of PCR fragments was incubated with the pGADT7-rec plasmid (Clontech) following the co-transformation protocol described in the “Matchmaker Library Construction & Screening User Manual (Clontech)”. Cells were plated on YSD selective media lacking Trp (W), Leu (L), Ade (A) and His (H) and supplemented with 100 μM IAA. Plasmids were recovered from those colonies able to grow in presence of IAA and sequenced.

Error-prone PCR mix was composed of G2 polymerase buffer (Promega) and G2 polymerase, 5 mM MgCl₂, 0.15 mM MnCl₂, and unbalanced dNTP mix (2.5 mM dATP, 2.5 mM dGTP, 10 mM dCTP, 10 mM dTTP). The pool of PCR fragments was incubated with the pGADT7-rec plasmid (Clontech) following the cotransformation protocol described in the Matchmaker library construction and screening user manual (Clontech). Cells were plated on YSD-selective medium lacking Trp (W), Leu (L), Ade (A), and His (H) and supplemented with 100 μM IAA. Plasmids were recovered from those colonies able to grow in the presence of IAA and sequenced (total of 27 colonies). One clone carried five mismatches, including the D30G mutation, and three had a deletion of 12 amino acids, including the D30 residue. The remaining 23 clones contained several errors that affected the correct frame and for this reason were discarded. The IND promoter (3.2 kb upstream of the ATG) plus the IND/IND^D30G-coding region were cloned into the pCGN1547 vector already harboring the tNOS terminator. The Agrobacterium tumefaciens strain GV3101 containing the final vector was transformed into ind-2 plants using the floral dip method.