BL21(DE3) pLysS Chemically Competent E. coli

Size: 100μl


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GenSunBio BL21(DE3) pLysS Competent E. coli is suitable for transformation and protein expression. This strain does not express the T7 RNA polymerase. GenSunBio BL21(DE3) pLysS Competent E. coli is an E. coli B/r strain and does not contain the lon protease. It is also deficient in the outer membrane protease, OmpT. The lack of these proteases reduces degradation of heterologous proteins expressed in this strain.

The pLysS plasmid carried by the GenSunBio BL21(DE3) pLysS Competent E. coli produces T7 lysozyme to reduce basal level expression of the gene of interest. The strain is recommended for use when expressing toxic genes. pLysS confers resistance to chloramphenicol (CamR) and contains the p15A origin. This origin allows pLysS to be compatible with plasmids containing the ColE1 or pMB1 origin (i.e. pUC- or pBR322-derived plasmids).

Genotype:F- ompT hsdSB (rB-mB-) gal dcm (DE3)pLysS (CamR)

Storage: -80

General Guidelines:

Follow these guidelines when using GenSunBio BL21(DE3) pLysS Chemically Competent E. coli.

• Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting. Thaw GenSunBio BL21(DE3) pLysS competent cells on ice, and transform cells immediately following thawing. After adding DNA, mix by swirling or tapping the tube gently. Do not mix cells by pipetting.

Transforming Competent Cells: 

Perform the following before starting the transformation procedure: 

• Equilibrate a water bath to 42

• Warm the SOC. Medium or LB Medium to room temperature. 

• Warm the selective plates in a 37 incubator for 30 minutes (use 1 or 2 plates for each transformation). If you are including the pUC19 control, make sure that you have one LB agar plate containing 100μg/mL ampicillin. 

Transformation Procedure:

Use this procedure to transform GenSunBio BL21(DE3) pLysS chemically competent E. coli. We recommend including the pUC19 control plasmid DNA in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.

1.      Thaw, on ice, one vial of GenSunBio BL21(DE3) pLysS chemically competent cells for each transformation. 

2.      Add 1 to 5 μl of the DNA (10 pg to 100 ng) into a vial of GenSunBio cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10 pg (1μl) of DNA into a separate vial of GenSunBio cells and mix gently.

3.      Incubate the vial(s) on ice for 30 minutes. 

4.       Heat-shock the cells for 90 seconds at 42 without shaking.  

5.       Remove the vial(s) from the 42bath and place them on ice for 2 minutes.

6.       Aseptically add 250μl of pre-warmed SOC or LB Medium to each vial. 

7.      Cap the vial(s) tightly and shake horizontally at 37 for 45 minutes at 150 rpm in a shaking incubator.

8.      Spread 20-200 μl from each transformation on a pre-warmed selective plate and incubate overnight at 37. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, dilute the transformation mix 1:10 into LB Medium (eg. remove 100 μl of the transformation mix and add to 900 μl of LB Medium) and plate 25-100 μl.

9.      Store the remaining transformation mix at +4°C. Additional cells may be plated out the next day, if desired.

10.    Invert the selective plate(s) and incubate at 37°C overnight.

11.    Select transformants from the plates and culture as expression.


• GenSunBio BL21(DE3)pLysS Chemically Competent E. coli should be stored at -80. Storage at -20 will result in a significant decrease in transformation efficiency. GenSunBio BL21(DE3)pLysS Chemically Competent E. coli lose efficiency whenever they are warmed above -80, even if they do not thaw.

•Clones may exhibit differences in expression of heterologous genes. We recommend choosing 3-4 transformants when characterizing clones for protein expression.

• GenSunBio BL21(DE3)pLysS cells require IPTG to induce expression of the T7 RNA polymerase from the lacUV5 promoter.

• GenSunBio BL21(DE3)pLysS Chemically Competent E. coli. are for research use only. Not intended for human or animal diagnostic or therapeutic uses.