Genelute minus etbr spin columns

To construct the glutathione-S-transferase (GST)-fused protein expression plasmids, the cDNA sequences were recombined into the pGEX-4T vector (GE Healthcare Life Sciences, Pittsburgh, PA, USA) as previously described (32 (link), 35 (link), 37 (link)). The pBluescript plasmids associated with the cDNA inserts were digested with the restriction endonucleases EcoRI and XhoI and detached by agarose gel electrophoresis. GenElute Minus EtBr spin columns (Sigma-Aldrich Corp., St. Louis, MO, USA) were used to isolate the cDNA fragments which were ligated in frame to EcoRI- and XhoI-digested pGEX-4T-3 linearized vectors with ligation convenience kits (Nippon Gene, Toyama, Japan). The ligation mixtures were used to transform ECOS-competent E. coli BL-21 cells (Nippon Gene). Successful recombination was confirmed by DNA sequencing and protein expression analysis.

Cloning and sequencing were performed for carrot only, to conclusively confirm Lso infection. Briefly, two carrot amplicons (from two symptomatic carrot plants collected in August and October 2010, respectively; see Results section) were selected and excised using clean razor blades and ethidium bromide was removed using GenElute Minus EtBr Spin columns (Sigma-Aldrich, Inc., St Louis. MO). The purified PCR products were cloned using the TOPO TA cloning kit® (Invitrogen, Carlsbad, CA) with TOP 10 Escherichia coli chemically competent cells. Plasmid DNA was extracted from selected colonies using the QIAprep spin mini prep kit (Qiagen, Valencia, CA) and the DNA clones were sequenced at MC Laboratories (MCLab, San Francisco, CA).

Expression plasmids of GST fusion proteins were constructed by recombining the cDNA sequences into pGEX-4T vectors (GE Healthcare Life Sciences, Pittsburgh, PA) as previously described [16 , 17 (link), 23 (link)-26 (link), 28 (link)-32 (link), 34 , 39 (link), 42 , 43 (link)]. The pBluescript plasmids containing cDNA inserts were digested with EcoRI and XhoI and separated via agarose gel electrophoresis. The inserted cDNA fragments were isolated using GenElute Minus EtBr Spin Columns (Sigma-Aldrich, St. Louis, MO). Using Ligation Convenience Kits (Nippon Gene, Toyama, Japan), the inserts were properly ligated in frame to EcoRI- and XhoI-digested pGEX-4T-1 or pGEX-4T-3 linearized vectors, which express the recombinant GST-tagged proteins. The ligation mixtures were used to transform ECOS competent E. coli BL-21 cells (Nippon Gene), and appropriate recombinants were confirmed by DNA sequence analysis as well as protein expression. To confirm successful recombination, expression of GST fusion proteins was induced by treating the transformed bacterial clones with 0.1 mM IPTG for 2.5 h, and expressed proteins were electrophoresed through 11% sodium dodecyl sulfate (SDS)-polyacrylamide gels.