Yeast and Insect DNA Purification Protocols

Yeast genomic DNA was purified by lysing cells by glass bead disruption, followed by phenol/chloroform extraction and ethanol precipitation (Hoffman and Winston, 1987 (link)). Wolbachia DNA was purified by homogenizing 10 whole infected insects in lysis buffer and recovering DNA with organic extraction following referenced protocols (Beckmann et al., 2017 (link); Beckmann and Fallon, 2012 (link)). Drosophila melanogaster and D. simulans lines infected with wMel, wRi, and wHa were used as PCR template sources. In some cases, genes from wNo and wMel were subcloned from synthesized constructs (Genscript). Genomic wStr DNA was a gift from Ann Fallon and was derived from infected cell cultures. PCR amplicons were produced with primers listed in Supplementary file 1i. High fidelity Phusion polymerase (New England Biolabs) was used to amplify DNA, which was then restriction enzyme digested, gel-purified and ligated into various plasmid vectors (Supplementary file 1j). Plasmids were sequenced and confirmed at the Yale Keck Foundation DNA sequencing facility. Point mutations were introduced by QuikChange site-directed mutagenesis (Stratagene). Other modifications such as truncations or tag additions/swaps were created by site-directed ligase-independent mutagenesis (SLIM) (Chiu et al., 2004 (link)).