Semi-random nested PCR

To map the transposon insertion sites, plaque-purified R. parkeri strains were boiled for 10 min and used as templates for PCR reactions. Genomic DNA at insertion sites was amplified for sequencing using semi-random nested PCR. The first “external” PCR reaction used transposon-specific primers (ExTn1 5’-CACCAATTGCTAAATTAGCTTTAGTTCC-3’; or ExTn2 5’-GTGAGCTATGAGAAAGCGCCACGC-3’) and a universal primer (Univ1 5’-GCTAGCGGCCGCACTAGTCGANNNNNNNNNNCTTCT-3’). Univ1 has a specific sequence at the 5’ end and a random sequence near the 3’ end to allow for random annealing throughout the chromosome. The first PCR reaction yielded the “external” product that served as a template in the subsequent “internal” PCR reaction using transposon-specific primers (InTn1 5’-GCTAGCGGCCGCGGTCCTTGTACTTGTTTATAATTATCATGAG-3’; or InTn2 5’-GCTAGCGGCCGCCCTGGTATCTTTATAGTCCTGTCGG-3’) and a different universal primer (Univ2 5’-GCTAGCGGCCGCACTAGTCGA-3’). Univ2 contains the same specific sequence as Univ1, allowing for specific amplification of the external PCR product. PCR products were cleaned using ExoSAP-IT PCR Product Cleanup Reagent (Affymetrix) and sequenced using transposon-specific primers SR095 5’-CGCCACCTCTGACTTGAGCGTCG-3’ and SR096 5’-CCATATGAAAACACTCCAAAAAAC-3’. Genomic locations were determined using BLAST against the R. parkeri strain Portsmouth genome (GenBank/NCBI accession {"type":"entrez-nucleotide","attrs":{"text":"NC_017044.1","term_id":"383483341","term_text":"NC_017044.1"}}NC_017044.1).