Protocol for the preparation of competent cells Andreas Leibbrandt Source: modified Hanahan procedure after Methods Enzymol. 1991; 204:63-113 buffers to render cells competent: DH5 FSB + 5% sucrose XL10-Gold FSB or TB TOP10 CCMB80 FSB buffer sucrose: 10 mM KOAc pH 7.5 100 mM KCl 45 mM MnCl2 10 mM CaCl2 3 mM HexCo(III)Cl 10% glycerol [5% sucrose] pH adjusted to 6.4
TB buffer: 10 mM PIPES pH 6.7 55 mM MnCl2 15 mM CaCl2 250 mM KCl
CCMB80 buffer: 80 mM CaCl2 20 mM MnCl2 10 mM MgCl2 10 mM KOAc pH 7.0 10% glycerol pH adjusted to 6.4 Material check list:
+ 500 ml of SOB or SOB medium (order from IMP media kitchen; add 6.25 ml of 1M MgCl 2 and + 6.25 ml of 1M MgSO4 prior to use, i.e. SOB ), use ~250 ml per E. coli strain o SOB: for CCMB80-competent cells, i.e. Mach1 and TOP10 cells o SOB+: for FSB-competent cells, i.e. DH5 and XL10-Gold cells o SOB+, 40 g/ml Cam, 80 g/ml Tet: for XL10-Gold cells 2 l Erlenmeyer flask with red lid (ask the IMP media kitchen to rinse and autoclave as for cell culture glassware) 0.5 l Erlenmeyer flask (ask the IMP media kitchen to rinse and autoclave as for cell culture glassware) 10 ml Falcon 2059 tube pre-cooled CCMB80, TB, or FSB buffers pre-cooled 1.5 ml Sarstedt screw cap tubes (from IMP store) pre-cooled Eppendorf Combitip 5 ml pre-cooled Falcon tubes, 50 ml pre-cooled serological pipettes (5 and 10 ml) ART200 tips Vortexer in the cold room
liquid N2 or EtOH/dry ice bath in the cold room ice basket(s) to incubate cells and transfer them from the centrifuge to cold-room DAY 1-2 pick 5 single colonies and resuspend by gentle vortexing in 1.5 ml of SOB(+) in a Falcon 2059 tube o e.g. from a 10-6 dilution prepared from a frozen stock of competent cells, plated on SOB (+) agar plates and grown o/n @ 37°C o alternatively, scrape off some cells from a frozen glycerol stock and resuspend by gentle vortexing in 1.5 ml of SOB(+) in a Flacon 2059 tube inoculate in 20 ml of SOB(+) in a 0.5 l Erlenmeyer flask and grow @ 18°C until the culture becomes turbid DAY 3-4
(+) on the next day, dilute 1:100 in fresh SOB medium and grow cells to an OD600 of ~0.35-0.6 @ 18°C o growth @18°C is very slow, so it might be best to start of ~noon the day before to finish the preparation on the next day transfer cells to 5 cooled 50 ml Falcon tubes, and incubate on ice for ~15-30' o optionally: prepare glycerol stock, i.e. cells 1:1 with 60%SOB, 40% glycerol spin down cells @ 4000 rpm for 15' at 4°C o don't forget to pre-cool the centrifuge and centrifuge containers in the cold room, decant the supernatant and keep the tubes inverted for several minutes to drain off excess media pool the cells (i.e. 5 Falcon tubes) by resuspending the cell pellets carefully (by gentle vortexing or pipetting) in 1/80-1/85 of the original volume in the respective buffer (i.e. for 250 ml of cells, use 3 ml of buffer) incubate bacteria on ice for 20' for FSB preparations, add 3.5% DMSO (105 µl DMSO/3 ml buffer/ 250 ml SOB (+)) from a freshly thawed aliquot of DMSO to cells, mix by gently swirling the tube and incubate on ice for 10' o DMSO is stored @ 20°C; remove an aliquot at the beginning of the procedure since it takes a while to defrost o apply DMSO drop by drop to the center of the solution and gently swirl the mixture add the same volume of DMSO as before, mix, and further incubate on ice for 5' aliquot 0.2 ml of DMSO-treated competent cells into pre-cooled 1.5 ml Eppendorf tubes and
shock-freeze competent cells in liquid N2 or an EtOH-dry ice bath, store cells @ 80°C o aliquot cells by using the Eppendorf Multipette with a pre-cooled 5 ml Combitip