One shot ccdb survival 2 t1

Liquid culture of the bacteria strain SURE2 (Agilent Technologies, cat. #200152) or One Shot ccdB Survival 2 T1 (Invitrogen, cat. #A10460) was set up from a single colony or from frozen glycerol stock in 20 ml of antibiotic-free Luria-Bertani (LB) medium and incubated at 37 °C in an incubator shaker (Infors, HT Multitron, cat. #2292113-18) at 200 rpm overnight. The next day, 10 ml of the bacteria culture was added to 100 ml LB and incubated at 37 °C in an incubator shaker at 200 rpm until an OD₆₀₀ = 0.48 was reached. The bacterial culture was chilled on ice for 20 min before centrifugation at 4 °C at 1000×g for 10 min. The supernatant was discarded, and bacterial pellets were resuspended and pooled in 30 ml ice-cold TFBI (100 mM rubidium chloride, 64.3 mM manganese (II) chloride, 30 mM potassium acetate, 10 mM calcium chloride, 15% glycerol, adjusted to pH 5.8). After incubation on ice for 90 min, bacterial suspension was centrifuged at 4 °C at 900 × g for 10 min. The supernatant was discarded, and the bacterial pellet was resuspended in 4 ml ice-cold TFBII (10 mM rubidium chloride, 14 mM MOPS, 75 mM calcium chloride, 15% glycerol, adjusted to pH 7.0). Chemo-competent bacteria were aliquoted and flash-frozen in liquid nitrogen before long-term storage at −80 °C.

Using a single colony or a frozen glycerol stock, a liquid culture of the bacterial strain One Shot ccdB Survival 2 T1 (Invitrogen, cat. #A10460) or MegaX DH10B T1 (Invitrogen, cat. #C640003) was set up in 100 ml antibiotic-free LB medium and incubated at 37 °C in an incubator shaker (Infors, HT Multitron, cat. #2292113-18) at 200 rpm overnight. The following day, 10 ml of the overnight culture was added to 200 ml pre-warmed LB medium and grown at 37 °C in an incubator shaker (Infors, HT Multitron, cat. #2292113-18) at 200 rpm until an OD₆₀₀ = 0.5 was reached. The bacterial suspension was chilled on ice for 20 min and centrifuged at 4 °C at 2200 × g for 15 min. The supernatant was discarded, and the bacterial pellet was resuspended in 200 ml ice-cold 10% glycerol. After centrifugation, the supernatant was discarded, and the bacterial pellet was resuspended in 50 ml ice-cold 10% glycerol. The bacterial suspension was spun-down, the supernatant was discarded, and the pellet was resuspended in 8 ml ice-cold 10% glycerol. After final centrifugation, the bacterial pellet was resuspended in 600 μl ice-cold 10% glycerol and directly used for electroporation or stored at −80 °C.