Preparation of electrocompetent cells \nWeinstock paper:Matthew T Weinstock,\u00a0Eric D Hesek,Christopher M Wilson,\u00a0Daniel G GibsonVibrio natriegens as a fast-growing host for molecular biology\nNature Methods volume\u00a013,\u00a0pages\u00a0849\u2013851\u00a0(2016)To prepare the day before:\n\n-LB I media +V2 salts (204 mM NaCl, 4.2 mM KCl, 23.14mM MgCl2 )-electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH 7) ( sterile filtrated)-over night culture of V.n from the cryo-stock in LBI + v2 salts (37 \u00b0C ; at 200 r.p.m)Preparing culture :Depending on how many aliquds you want to have, incubate media with your overnight culture for a starting OD of 0.05.The culture is grown at 37 \u00b0C in a baffled flask, shaking at 200 r.p.m. until an OD600 between 0.5 to 0.8 is reached.\n*be careful when they reach an OD near 0.1, V. natriegensis very fast growing, so start measuring in shorter time periods.Prechill the electroporation bufferWashing:From here on try always to keep your culture on ice.-The culture is then put on ice for 15 min. ( the original Protocol sais to directly fill them into your prechilled centrifugatin containments )-The cells are pelleted at 3000x g. for 20 min at 4 \u00b0C.-The supernatant is carefully decanted and the cell pellets are gently suspended in 10mL\n of chilled electroporation buffer.-The suspensions are transferred to a chilled50mL falcon tube and the tube is filled top\n with additional chilled electroporation buffer (50mL) and inverted several times. -The cells are centrifuged down at 3000x g for 15 min at 4 \u00b0C. -The wash is repeated two times for a total of three washes.Aliquotation:After the final wash, the supernant is carefully decanted the cells are gently resuspended in residual electroporation buffer. \nMeasure the OD in a 1\/20 dilution against electroporation buffer.The volume is adjusted with additional electroporation buffer to bring the final OD600 to 16.The Cells are aliquoted ( 80\u00b5L ) into chilled 1.5\u00b5L centrifugation tubes, directly frozen in liquid nitrogen and stored at \u221280 \u00b0C until use.