NGS of E. coli : genomic amplicon
On average, ~8000 colonies were washed from CAST-directed integration experiment plates and cultured in 4 mL selective LB. 1 mL of the culture was aliquoted for genomic DNA extraction using the Qiagen DNeasy Blood & Tissue Kit. 5 ng of the extraction for each genomic target site (L02, L09, L14, L21) was amplified with the PrimeSTAR GXL DNA Polymerase in 50 L reactions each. Amplification primers were designed to bind ~200bp away from each integration site to capture the insertion junction. X L of the PCR reaction was purified with the Qiagen QIAquick PCR Purification Kit and sent to the sequencing core at the California Institute of Technology (Millard and Muriel Jacobs Genetics and Genomics Laboratory). Samples were Illumina sequenced at 50bp resolution.