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URL: https://dx.doi.org/10.17504/protocols.io.u3geyjw

Authors: Josef Hoff

Summary: Electroporation protocol Weinstock paper:Matthew T Weinstock, Eric D Hesek,Christopher M Wilson, Daniel G GibsonVibrio natriegens as a fast-growing host for molecular biology Nature Methods volume 13, pages 849–851 (2016)To prepare before:recovery medium(BHI + v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14mM MgCl2), and 680 mM sucrose) sterile filtration * i have a box of aliquots in the freezer and pre heat them before use-A vial of competent cells is retrieved from storage at −80 °C and allowed to thaw on ice.-Plasmid DNA and electrocompetent cells are combined and gently mixed in a chilled 1.5-mL microcentrifuge tube. -The cell–DNA suspension is transferred to a chilledelectroporation cuvette with a 0.1-cm gap size.-Cells are electroporated with 900V (in our Electroportor we cant set other parameters ) * in the Weinstock paper they recommend depending on the strain 700-900V, 25 μF and 200 Ω-Cells are immediatelyrecovered in 500 μL preheated (50°C) recovery medium and transferred to a 1,5-mL tube. * we preheat the media to 50°C because the recovery media is cooled down by pipetting and up taking the chilled cells from the cold cuvettes-The cells are recovered by incubating at 37 °C for 1.5h. ( also put the agar plates for preheating in the incubator at 37°C )-The cells are centrifuged down for one mintute at 3000g. The supernant is then discanted.- the pellet is resuspendet in the leftover oft he media and plated out on warm agar plates containing appropriate antibiotic. * for Chloramphenicol 2µg/mL ; for Kanamycine 200µg/mL; for Carbenicillin 200µg/mL-The plates are incubated for several hours or overnight at 37 °C for colonies to appear.

Affiliations: Phillips University

Version: 1

Publication Date: 2018

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