Preparation of electrocompetent cells v1

Preparation of electrocompetent cells Weinstock paper: Matthew T Weinstock,Eric D Hesek,Christopher M Wilson,Daniel G Gibson Vibrio natriegens as a fast-growing host for molecular biologyNature Methods volume13,pages849–851(2016) To prepare the day before: -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 °C ; 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 °C in a baffled flask, shaking at 200 r.p.m. until an OD600 between 0.5 to 0.8 is reached.*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 buffer Washing: 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 °C. -The supernatant is carefully decanted and the cell pellets are gently suspended in 10mL of chilled electroporation buffer. -The suspensions are transferred to a chilled50mL falcon tube and the tube is filled top with additional chilled electroporation buffer (50mL) and inverted several times. -The cells are centrifuged down at 3000x g for 15 min at 4 °C. -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. Measure 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 ( 80µL ) into chilled 1.5µL centrifugation tubes, directly frozen in liquid nitrogen and stored at −80 °C until use.