Genome sequencing has been accelerated forward by the use of cloning large fragments of genomic DNA into fosmids. These cloned pieces of DNA are usually up to 40 kb in size, whereas the fosmid vectors themselves are predominantly around 9 kb. Despite the challenges of low vector copy number, the need for high throughput automation of fosmid template preparation, like BAC’s, is significant. Here we describe a semi-automated preparation method using the Montage BAC96 Miniprep Kit on the Biomek FX workstation. Vacuum filtration and shaker mixing steps were performed off-line, however, these can be carried out on the FX utilizing SPE and orbital shaker ALP’s, thus supporting a fully automated procedure.
The protocol was developed to maximize yields, decrease well-to-well variability, and provide high quality DNA sequence data. Culture and lysis conditions were optimized to accomplish this. The protocol, which relies primarily on vacuum filtration for sample processing, takes approximately 1 hour. |
