Blommel, P.G., Martin, P.A., Wrobel, R.L., Steffen, E. and Fox, B.G.
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)