PRODUCT INFORMATION

DynaCompetent Cells

Product Name :BL21(DE3) for Electroporation(Previous name: Electrocompetent Cell BL21(DE3))
Code No. :DS258

Size :

50 μl × 5

Competency :> 2 × 109 cfu/μg (50 μl cell, pUC19)

Supplied product :SOC medium, 1 ml × 5

This product is for research use onlyDescription :DynaCompetent Cells

BL21(DE3) for Electroporation is designed for high efficiency transformation byelectroporation and is suitable for DNA library construction and all kinds of cloning procedures. E. coli strain
BL21(DE3)

is a widely used strain for bacterial protein expression. The BL21(DE3) strain contains theλDE3 lysogen, which expresses T7 RNA polymerase under the control of the lacUV5 promoter1). Upon
addition of IPTG, T7 RNA polymerase is expressed and it induces a high-level protein expression from T7
promoter driven expression vectors (e.g., pET).
F– ompT hsdS(rB– mB–) gal dcm λ(DE3)

Genotype of E. coli strain BL21(DE3) :( λ(DE3): lacI, lacUV5-T7 gene 1, ind1, sam7, nin5 )Quality control :Transformation by electroporation was carried out according to the method described in this Product
Information using supercoiled pUC19 plasmid. Transformants were plated on LB plates containing 50 μg/ml
ampicillin. The efficiency was confirmed to be greater than 2 × 109 cfu/μg with 50 μl of the cells.
Storage condition :Stable at -80℃ with little or no loss in transformation efficiency for 12 months from the date of receipt.
Upon receipt, store the DynaCompetent Cells BL21(DE3) for Electroporation in a freezer at -80oC directly from a
dry ice shipping box and store SOC medium at room temperature or at -80oC.
Handling of competent cells :• After thawing electrocompetent cells on ice, cells tend to lose transformation efficiency gradually.
Transformation should be started immediately following thawing cells on ice.
• Electroporation is temperature-dependant. Electrocompetent cells should be maintained at 0-4°C before
pulsing with electroporation apparatus.
• Use of refrozen electrocompetent cells is not recommended.
Composition of SOC medium supplied :20 g/L5 g/L
0.5 g/L
0.186 g/L
2.4 g/L
4 g/L

tryptoneyeast extract
NaClKCl
MgSO4, anhydrousglucose

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PRODUCT INFORMATION
Transformation (electroporation) procedure :● Materials to be supplied by user
• Electroporation apparatus• Sterile electroporation cuvettes (0.1 cm gap)

• LB plates with antibiotic• Sterilized pasteur pipettes• Sterile 14 ml polypropylene culture tubes• 37°C shaker• Sterile spreaders• 37°C incubator● TransformationBefore start:• Chill sterile electroporation cuvettes (0.1 cm gap) on ice.
* Electrocompetent cells should be maintained at 0-4°C before pulsing.
• Place 14 ml sterile polypropylene culture tubes containing 950 μl of SOC medium (at roomtemperature) for each electroporation near the electroporation apparatus.
* It is important to add SOC medium to electrocompetent cells immediately after pulsing.
• Set up the electroporation apparatus according to the manufacturer’s instructions.
1. Thaw the electrocompetent cells on ice and add DNA solution*(1-3 μl) to the cells then flick gently.
* DNA solution*: Highly purified DNA is recommended. DNA should be dissolved in pure water or
1×TE. For ligation mixture, see “To use ligation mixture for electroporation” below.
2. Quickly transfer the mixture of cells and DNA to a chilled electroporation cuvette, and tap the mixture
to the bottom of the cuvette.
* Electroporation should be performed immediately after mixing electrocompetent cells and DNA.
3. Place the cuvette into the electroporation apparatus, then pulse.
4. Remove the cuvette from the electroporation apparatus and immediately add the 950 μl of SOC
medium to the cuvette with a sterile pasteur pipette to suspend cells.
5. Transfer the cell suspension to the sterile 14 ml polypropylene culture tube. Culture the cell at 37°C
for 1 hr in a shaker.
6. Spread aliquot of the cell to an LB agar plate containing appropriate antibiotic.
7. Incubate the plate at 37°C overnight.

● To use ligation mixture for electroporation
Ligation mixture contains salts and impurities that interfere with electroporation. To use ligation mixture forelectroporation, DNA should be purified by phenol/chloroform extraction following ethanol precipitation orwith a spin column. The phenol/chloroform extraction following ethanol precipitation gives successive
results for ligation mixture prepared with DNA Ligation Kit ver. 2 (DS110).

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PRODUCT INFORMATION● Notes for transformation
1. As T7 expression method is a high-level protein expression system, some basal level expression of thetarget protein will occur in uninduced cells. This is likely to be problematic in cases in which the target
protein is toxic to E. coli. cells. In this case, it may be necessary to decrease the basal level expression asfollows:
1) Use a low-copy, T7 driven expression vector.
2) Use liquid medium and agar plates supplemented with glucose (0.5 -1 %).
Glucose is known to decrease a basal expression from lacUV5 promoter2).
2. Expression may vary among transformants. If large and small colonies are observed in the same plate,the expressed protein may affect the growth of the E. coli cells.
3. If the expressed protein is toxic to E. coli cells, transformants may not be obtained.

Protein expression procedure :The following protocol is a general guide for the protein expression by use of T7 promoter driven expression
vectors and BL21(DE3) cells.

Before starting:
1. Construct T7-promoter expression plasmid harboring a gene of interest using non-expression hosts.
2. Transform DynaCompetent Cells BL21(DE3) for Electroporation with the expression plasmid.

Expression:
1. Following electroporation, pick a colony and inoculate it into 3 ml of LB medium containing theappropriate antibiotic with shaking at 37°C, overnight.
2. The next morning, transfer 0.5 ml of the overnight culture to a new 10 ml of LB medium containing theappropriate antibiotic to select the expression plasmid. Grow the culture with shaking at 37°C until theOD600 reaches 0.5 (approximately 2 hrs but this depends on the expression plasmids).
3. When the OD600 reaches 0.5, add IPTG to a final concentration of 1 mM to the rest of the culture andgrow the culture with shaking at 37°C for 3 hours. The IPTG concentration and induction time aregeneral values. It is recommended to determine the optimal condition for the target gene expression.
4. After the induction, harvest the cells.

3Ver. 3.0

PRODUCT INFORMATION
Reference:1) Studier, F.W. and Moffatt, B.A., J. Mol. Biol. 189 (1986) 113–130.
2) Pan, S. and Malcom, B.A., BioTechniques 29 (2000), 1234–1238.

General reference in this Product Information
Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY.

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BL21(DE3)pLysS● Purchaser Notification
This product is manufactured based on the T7 expression system which is the subject of US patentapplications assigned to Brookhaven Science Associates, LLC (BSA). The product must be used only outside
the United Stated and its territories. Neither this product nor materials prepared used by the T7 expression
system are allowed to be distributed in the US and its territories without license of BSA. Information about
license regarding the T7 expression system maybe obtained from the Office of Intellectual Property andSponsored Research, Brookhaven National Laboratory, Building 185, P.O. Box 500, Upton, New York
11973-5000, USA.

4Ver. 3.0