title: GSM3415095: rep1 50mM MgSO4 gate 6 [degenerate_library_sort_seq]; Escherichia coli str. K-12 substr. MG1655; OTHER
library_construction_protocol: plasmid DNA was extracted with the Quiagen plasmid Miniprep Kit [degenerate_library_sort_seq] For the two mutagenized regions of the plasmid to be brought into close enough proximity (< 790 bp) for paired-end Illumina sequencing, plasmids were digested with XhoI and then self-ligated (T4 ligase, 4 hr). To isolate only self-ligation products, and not cross-ligation products, ligation reactions were cleaned (Zymo PCR Clean Up) and gel purified to select for the correct size on FlashGels (Lonza). Two PCR reactions were performed, both using KAPA HiFi Hotstart, to add Illumina sequencing adaptors and barcodes. First, ligation reaction products were amplified for 30 cycles (95 °C for 30 s, 65 °C for 15 s, 72 °C for 120 s) with primers CJM642 and CJM643 in an emulsion PCR (Micellula Emulsion PCR) to avoid PCR chimeras. Second, purified PCR product from the first reaction was subjected to a second PCR with barcoding primers for 9 cycles (95 °C for 30 s, 65 °C for 15 s, 72 °C for 60 s). Final products were quantified (NanoDrop), normalized, combined, and sequenced on an Illumina NextSeq.  [degenerate_library_sort_seq]
attributes.tag: GEO Accession
attributes.value: GSM3415095