| description |
These 4,096 clones were put into a 16X16X16 cube and assigned to 96 pools of 6 types using the solid pooling strategy. Each pool contained 16X16=256 clones. Clones were picked into 96-well culture plates containing 1,000 μl freezing media in each well, cultured for 18 hours at 37°C, and then stored at -80°C. In each 96-well culture plate, 8 successive clones in columns or rows were combined into a primary pool. If there were not 8 clones in a column or row of the plate, then clones from the next plate were mixed into the primary pool. Each primary pool contained 8X 150 μl of each well (total 1,200 μl) and was stored in another 96-well culture plate. According to the pool design, every 32 primary pools were replicated to a new 96-well culture plate, and each well contained 100 μl of liquid from the primary pool and 1,100 μl 2×YT media. The primary pools stored in the wells from columns 1 to 4 formed a pool block, and those stored in the wells from columns 9 to 12 formed another pool block. Therefore, each pool plate stored two pool blocks, each of which contained 256 clones. Pool plates were cultured for 18 hours at 37°C. Finally, each pool block was replicated for 15 copies by mixing 40 μl of the liquid of each pool block well with 1,000 μl 2×YT media; the 15 copies were then cultured for 18 hours at 37°C and used to extract plasmid DNA using the QIAGEN? Large-Construct Kit. Plasmid DNAs of all 96 pools were paired-end sequenced by Illumina Genome Analyzer. The DNA fragments in the sequencing library were approximately 500 bp in length. Each read was 100 bp; the accumulated read length of each pool was more than 109 bp, representing a pool coverage of more than 30X. |