Preparation of DH5 and XL10-Gold Competent Cells and Presumably Also TOP10 Cells (CCMB80

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