BLR(DE3)pLysS 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 made of Escherichia coli BLR(DE3)pLysS. This strain is a derivative strain of the BLR(DE3) strain，and the recA mutation favors the stabilization of the prokaryotic expression plasmid (suitable for protein expression of genes containing unstable DNA or repetitive DNA sequences). The chromosome of this strain integrates the lambda phage DE3 region (the 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 protein expression of pET series，pGEX，pMAL and other plasmids. The pLysS plasmid carried by this strain contains a gene expressing T7 lysozyme，which can reduce the background expression level of the target gene，but does not interfere with the expression induced by IPTG，and is suitable for the expression of toxic and non-toxic proteins. BLR(DE3)pLysS strains are tetracycline，chloramphenicol resistant. BLR(DE3) pLysS competent cells were fabricated by a special process，and the transformation efficiency of pUC19 plasmid detection was > 1×108cfu/μg DNA. Genotype: F-ompThsdSB(rB-mB-) gal dcm (DE3)Δ(srl-recA)306::Tn10 pLysS(CamR，TetR) Procedure: 1. Remove competent cells from the -80℃ freezer，place them in 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.