Notes: The authors use the Flexi^® Cloning System to convert their cDNA clones to expression-ready clones. They wanted clones that could be used for comprehensive analysis with the HaloTag^® Technology. They also describe a method of transferring ORFs between Flexi^® Vectors in a 96-well plate format. They also used Wizard^® SV 96 Plasmid DNA Purification, Wizard^® SV PCR Clean-Up, and Wizard^® SV Gel and PCR Clean-Up Systems. (4056)

Notes: These authors used the Flexi^® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag^® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi^® System and Gateway^® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag^® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

Notes: In this study, the Flexi^® Vector Systems was compared with the Gateway^® Cloning System to determine its utility in high-throughput expression cloning by subcloning 96 human target genes. A direct comparison between pVP16, the Gateway^® vector and the equivalent Flexi^® Vector, pVP33A or K, was achieved by modifying pVP16 with the barnase gene and PmeI/SgfI restriction sites, duplicating the design available in the commercial Flexi^® Vectors. Capture of genes by PCR amplification of the cDNAs was similar for both systems, but the timeline for the Flexi^® Vector system was shorter at 6–8 days compared to 12 days for the Gateway^® system. They also found the Flexi^® Vector System was lower cost and more accurate due to the shorter primers required for the Flexi^® Vector cloning. The authors found nearly twofold fewer missense errors due to the shorter amplification primers. Ninty-six cDNAs were amplified simultaneously in their protocol and PCR products were cleaned up using either the MagneSil^® PCR Clean-Up System or Wizard^® SV 96 PCR Clean-Up, ligated into the Flexi^® Vector, and transformed into Select96™ Competent Cells. The study also compared transfer of cDNA inserts between different Flexi^® Vectors and transfer of cDNA inserts between different Gateway^® vectors and found similar performance in the two systems. For the Flexi^® Vector test set, the authors sequenced the clones, validating the high fidelity transfer of cDNA inserts between Flexi^® Vectors. (3533)

Notes: To prepare expression-ready cDNA clones of 1589 putative full-length ORFs (from human and mouse genes) with an average size of 2.8 kb, a linear trap vector was created. Generated by PCR, this linear plasmid contained gene-specific sequences to allow homologous recombination. In addition, 5–10µg of a plasmid containing the same gene-specific sequence and the linear trap vector were purified using the Wizard^® SV 96 PCR Clean-Up System. The purified DNA was resuspended in water and transformed into E. coli cells. The plasmid purified after the recombination was transferred to a Gateway expression vector and 100–200ng expressed in the TNT^® T7 Quick Coupled Reticulocyte Lysate System with 0.2µl of FluoroTect™ Green_Lys tRNA. The proteins expressed were resolved using SDS-PAGE. (3439)