Origami2(DE3) Competent Cells

1. Competent cells should be used immediately after thawing and should not be left in ice for too long.
2. Do not aspirate competent cells with a pipette，just flick your finger to mix well.
3. When inducing protein expression，the IPTG concentration is optional (0.1-2 mM). This product is a competent product made of Escherichia coli Origami2 (DE3). This strain is a K-12-derived strain containing mutated thioredoxin reductase (trxB) and glutathione reductase (gor) genes，which are two key enzymes in the reduction pathway，and their mutations favor the correct folding of dithiosein-containing proteins，enhancing the solubility of the proteins; At the same time，this strain is not kanamycin resistant and can be used for protein expression of kanamycin-resistant plasmids. The chromosome of Origami2 (DE3) strain integrates the lambda phage DE3 region (DE3 region contains T7 bacteriophage 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 the protein expression of pET series，pGEX，pMAL and other plasmids，and the Origami2 (DE3) strain has streptomycin and tetracycline resistance，and is a leucine growth-deficient strain. Origami2 (DE3) competent cells were fabricated by a special process，and the transformation efficiency of pUC19 plasmid detection was > 106 cfu/μg DNA. Genotype: Δ(ara-leu)7697ΔlacX74ΔphoAPvuII phoR araD139 ahpC galE galK rpsL F′[lac+ lacIq pro](DE3) gor522::Tn10 trxB (StrR，TetR) Procedure: 1. Remove competent cells from the -80℃ freezer and place on ice for 5 min. Add the plasmid of interest or ligation product，flick to mix evenly，and incubate in ice for 30 min; 2. Put it in a 42°C water bath for 90s，immediately insert it into ice and let it stand for 2-3min; 3. Add 900 μL of LB liquid medium and incubate at 220 rpm at 37℃ for 60 min; 4. Centrifuge at 5000 rpm for one minute to collect bacteria，leave about 100 μl of supernatant and gently pipette the resuspended bacterial block and apply it to a 2YT or LB plate containing the corresponding antibiotic，and place in an incubator at 37°C overnight.