Genejetplasmid miniprep kit

Immediately after sorting, 200 μL
of 1H,1H,2H,2H-perfluorooctanol (PFO) (Alfa Aesar) was added to the ∼150
μL double emulsions in nuclease-free water, vortexed, and centrifuged
quickly for 10 s. The top layer was extracted and added to a DNA-low
binding tube (Eppendorf). To the tube was added 4 μL of UltraPure
Salmon Sperm DNA solution (Thermo Fisher) diluted 100× in nuclease-free
water (final 2500× dilution) was added. The leftover PFO with
small amounts of aqueous phase on top was extracted once with a 100
μL solution of UltraPure Salmon Sperm DNA solution (Thermo Fisher),
diluted 2500x in nuclease-free water. To the 200 μL recovered
DNA, 1000 μL of DNA binding buffer (Zymo) was added and purified
over silica columns (Zymoclean Gel DNA Recovery, Zymo Research), eluting
in minimal amounts of nuclease-free water. The resulting purified
plasmids were transformed into E. cloni 10F ELITE Electrocompetent cells (Lucigen) and plated on two 140
mm Petri dishes containing LB_kan-agar. The next day, the
colonies were scraped, and the plasmid was isolated using a Genejet
plasmid miniprep kit (Qiagen). This purified plasmid stock was used
for transformation to BL21 (DE3) competent E. coli (NEB, 2527) for rescreening in plates.

Standard protocols were used for restriction endonuclease digestions, ligations, transformations and agarose gel electrophoresis, as previously described [55 ]. Plasmid DNA was extracted from E. coli using the Fermentas GeneJET Plasmid Miniprep Kit or the Qiagen Plasmid Midi Kit according to protocols provided by the manufacturers. Chromosomal DNA was extracted from P. aeruginosa using the Qiagen DNeasy Blood & Tissue Kit according to a protocol provided by the manufacturer. PCR products and restriction endonuclease digest products requiring purification were purified using the Promega Wizard SV Gel and PCR Clean-Up System according to a protocol provided by the manufacturer. Plasmid DNA was introduced into CaCl₂-competent E. coli cells, which were prepared as previously described [55 ]. Oligonucleotide synthesis was performed by Integrated DNA Technologies (Coralville, Iowa), and nucleotide sequencing was performed by ACGT Corporation (Toronto, Canada).