BLR(DE3) Competent Cells

1. Competent cells should be slowly thawed in ice，inserted into ice within 8 minutes to add target DNA，not placed in ice for too long，long-term storage will reduce the transformation efficiency.
2. Transformation of plasmids with high concentrations can correspondingly reduce the amount of bacteria that are ultimately used to coat the plate.
3. During induction，the IPTG concentration is optional (0.1-2 mM).
4. In order to obtain the required amount of protein，the optimal induction time，temperature，and IPTG concentration need to be optimized by the experimenter.
5. BLR(DE3) strains are tetracycline-resistant and cannot be used for transformation of tetracycline-resistant plasmids. BLR(DE3) is a derived strain of BL21(DE3) strain，and recA mutations favor the stabilization of prokaryotic expression plasmids (suitable for protein expression of genes containing labile DNA or repetitive DNA sequences). The chromosome of this strain integrates the lambda phage DE3 region (the DE3 region contains T7 phage RNA polymerase，which is integrated on the chromosome of E. coli)，and can simultaneously express T7 RNA polymerase and E. coli RNA polymerase，which is used for protein expression of pET series，pGEX，pMAL and other plasmids. The BLR(DE3) strain is tetracycline resistant. BLR(DE3) competent cells were fabricated by a special process，and the transformation efficiency of the pUC19 plasmid (2686 bp，AmpR) was > 1×108 cfu/μg DNA. Genotype: Operation method: 1. BLR(DE3) competent cells are taken out from -80℃，quickly inserted into ice，after 5 minutes，wait for the bacterial block to melt，add the target plasmid，and gently mix with the bottom of the EP tube by hand (avoid sucking with a gun)，and let stand in ice for 25 minutes. 2. Heat shock in a water bath at 2. 42°C for 45 seconds，quickly put it back into ice and let it stand for 2 minutes，shaking will reduce the conversion efficiency. 3. Add 700 μl of sterile medium (2YT or LB) without antibiotics to the centrifuge tube，mix well，and recover at 37°C for 60 min at 200 rpm. Centrifuge at 4000 rpm for one minute to collect the bacteria，leave about 100 μl of supernatant，gently pipette the resuspended bacterial blocks，and apply to 2YT or LB medium containing the corresponding antibiotic. 5. Place the plate upside down in a 37℃ incubator overnight.