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B2935
e coli protein expression
for protein expression and DNA plasmid production
Synonym(s):
BL21 Competent Cells, BL21 (DE3)T1R Competent Cells
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About This Item
MDL number:
UNSPSC Code:
12352200
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grade
Molecular Biology
for molecular biology
form
suspension
shipped in
dry ice
storage temp.
−70°C
General description
BL21(DE3)-T1R are competent E. coli that are suitable for high level induction and expression genes regulated by expression vectors with T7 promoter. The cells have transformation efficiency of >1x107 cfu/μg when transformed with non-saturating amounts of pUC19 plasmid DNA.
Sigma′s BL21-T1R competent E. coli cells are grown and made chemically competent using an optimized procedure specific to the strain, followed by strain verification and efficiency testing. The cells are provided in frozen 50 μl aliquots for convenience. Each aliquot can be used for a single transformation.
Application
Suitable for induction and expression of genes directed by the expression systems with T7 promoter
Features and Benefits
- Ensures induction and expression of genes from any T7-promoter based expression system
- Contains the genotype tonA that protects the clonal stocks from lytic bacteriophages
- Contains T7 RNA polymerase that is inducible by addition of IPTG to the culture
- Guaranteed high transformation efficiency
- Convenient 50 μL aliquots
Components
- BL21(DE3)-T1R chemically competent cells, 10 X 50 μL (B3060)
- pUC 19 control DNA (10 ng/μL), 10 μL (D2567)
Principle
BL21(DE3)-T1R does not express ion proteases and outer membrane protease, ompT. This prevents the degradation of heterologous proteins expressed by T7 expression vector systems. This strain also contains tonA genotype that confers resistance to lytic bacteriophages such as T1 and T5. The strain is lysogenic for lambda prophage and contains an inducible T7 RNA polymerase regulated by lacUV5 promoter.
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10 - Combustible liquids
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Pär Comstedt et al.
PloS one, 9(11), e113294-e113294 (2014-11-20)
There is currently no Lyme borreliosis vaccine available for humans, although it has been shown that the disease can be prevented by immunization with an OspA-based vaccine (LYMErix). Outer surface protein A (OspA) is one of the dominant antigens expressed
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What is the Department of Transportation shipping information for this product?
1 answer-
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Can I grow more competent cells from the stock I purchased?
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Generally, no, because the future generations of cells lose their competency.
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Competent cells were left on ice overnight - can they still be used?
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Competent cells left on ice, or allowed to come to room temperature slowly will suffer a dramatic loss in transformation efficiency. We recommend thawing a new aliquot of competent cells.
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Can I express a recombinant protein that is toxic to the bacteria?
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For expression of toxic recombinant proteins, select the BL21 strain containing the pLysS plasmid. These cells express low levels of lysozyme that will bind to T7 polymerase, and inhibit transcription.
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Are home-made competent cells as efficient at transformation as purchased cells?
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They can be, depending on the technique used, the expected transformation efficiency and how the cells are handled. For special applications, competent cells prepared in-house using standard methods may not provide the efficiency you need.
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What are the differences among the BL21 strains?
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The BL21-T1R cells are suitable for high level production of heterologous proteins directed by various expression vector systems (promoters such as lac, trc, tac, ?PL and araD). The BL21 (DE3) - T1R cells are suitable for high level induction and expression of genes from any T7 promoter-based expression vector. DE(3) indicates that the strain is lysogenic for a lambda prophage containing an inducible T7 RNA polymerase under control of the lacUV5 promoter.The BL21 (DE3) pLysS - T1R cells are suitable for high level induction and expression of genes from any T7 promoter-based expression vector pLysS. The cells express T7 lysozyme, a natural inhibitor of T7 polymerase, allowing for improved transcriptional control and reduction of "leaky" expression. The pLysS plasmid also renders the cell resistant to chloramphenicol (CmR) and contains the p15A origin. This allows pLysS to be compatible with plasmids containing the ColE1 or pMB1 origin.
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Can I re-freeze the vial if I do not use the entire aliquot of competent cells?
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Re-freezing competent cells will result in a decrease in their transformation efficiency. If the cells are frozen first in a dry ice / ethanol bath before placement in the -80C freezer, the loss will be about two-fold. If placed directly in the -80C freezer, the loss in transformation efficiency is about five to ten-fold.
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When using the competent cells for protein expression, what traits are required?
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Ideally, expression strains will minimally have the following genotype:EndA (DNA is not degraded)hsd (unmethylated DNA is not restricted)laclq (better control over the lac promoter)lon (lacks intracellular protease)ompT (lacks extracellular protease)pLysS (inhibits T7 RNA polymerase)tonA (resistant to T1 phage)trfA (replication of oriV plasmids)T7 RNA polymerase
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I’m working with BL21(DE3) pLysS and I’m getting a lot of background expression. Why?
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When using the BL21 (DE3) pLysS - T1R competent cells, selection should be performed for the introduced plasmid as well as the pLysS. This should be done by placing chloramphenicol (Cm) in selective plates and growth media. If there is still alot of background when selecting with Cm, switch to a promoter system that has better control.
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What DNA purity do I need for use in transformation?
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The DNA used should be high quality - free from phenol, proteins, detergents, and ethanol. Dissolve the DNA in sterile water or 0.5x TE buffer (5 mM Tris-HCl, 0.5 mM EDTA).
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