{"product_id":"neb-c2529h","title":"New England Biolabs, C2529H, NiCo21(DE3) Competent E. coli","description":"Having trouble opening your tubes?  \u003cb\u003eRelated Categories\u003c\/b\u003e Nickel Purification (His-tag),, T7 Expression,, Affinity Purification,  \u003cb\u003eApplications\u003c\/b\u003e His-tagged Protein Expression \u0026amp; Purification,, Affinity Purification \u0026amp; Expression Tags,, T7 Expression,  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eAntibiotic for Plasmid Selection\u003c\/b\u003e Antibiotics for Plasmid Selection Working Concentration Ampicillin 100 µg\/ml Carbenicillin 100 µg\/ml Chloramphenicol 33 µg\/ml Kanamycin 30 µg\/ml Streptomycin 25 µg\/ml Tetracycline 15 µg\/ml  \u003cb\u003eShipping Notes\u003c\/b\u003e Ships on dry ice  \u003cb\u003eFAQ\u003c\/b\u003e Q: Does New England Biolabs offer strains of Competent E.coli suited for protein expression? A: Yes, we have several strains for protein expression. BL21(DE3) (NEB #C2527) is offered for routine T7 Expression and BL21 (NEB #C2530) for non-T7 Expression. NEBExpress (NEB #C2523) is an enhanced BL21 derivative and NEB Express Iq (NEB #C3037) offers tightly controlled expression from vectors with lac, tac, trc, or T5-lac0 promoters. T7 Express Competent E.coli (NEB #C2566) and T7 Express Iq Competent E. coli (NEB #C3016) are enhanced derivatives of BL21(DE3). The lacIq strain allows for tighter control of expression. Both are available with the lysY feature for exceptional control of expression. (T7 Express lysY, NEB #C3010 and T7 Express lysY\/Iq, NEB #C3013). NEB also offers SHuffle™ strains that are capable of correctly folding proteins with multiple disulfide bonds in the cytoplasm. SHuffle™ Express (NEB #C3028H) can be used with non-T7 promoters or SHuffle™ Express T7 (NEB #C3029H) for T7 promoter expression vectors. For toxic T7 promoter expression, SHuffle™ Express T7 lysY (NEB #C3030H) is ideal. These 3 strains are also offered as K12 strains; SHuffle™ (NEB #C3025H), SHuffle™ T7 (NEB #C3026H) and SHuffle™ T7 lysY (NEB #C3027H). Lemo21(DE3) (NEB #C2528) is available for tunable expression of difficult targets such as membrane proteins, toxic proteins and proteins prone to insoluble expression. NiCo21(DE3) (NEB #C2529) is designed for routine expression and purification of His-tagged proteins. Q: What are the solutions\/recipes (C2529)? A: SOB: 2% Vegetable peptone (or Tryptone) 0.5% Yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4SOC:SOB + 20 mM GlucoseLB agar: 1% Tryptone 0.5% Yeast Extract0.17 M NaCl1.5% Agar Q: Why are there no colonies or no growth in liquid culture (C2529)? A: This outcome is often due to basal expression of a target gene product, which is detrimental to cell viability. Basal expression of target protein in NiCo21(DE3) is identical to basal expression in BL21(DE3). Some vector systems (in particular those utilizing a T7 promoter) allow expression without inducer. If tightly regulated T7 expression is required, use a strain expressing lysY: T7 Express lysY (NEB #C3010) lysY produces a mutant T7 lysozyme which binds T7 RNA polymerase, reducing basal expression of the target protein. Upon induction, newly made T7 RNA polymerase titrates out the lysozyme and results in expression of the target protein. T7 Express lysY\/Iq (NEB #C3013) lysY expression as well as lacI over-expression to repress basal expression of the T7 RNA polymerase. Lemo21(DE3) (NEB #C2528) BL21(DE3) containing the Lemo System™. LysY expression is modulated by L-rhamnose, making T7 protein expression tightly regulated and tunable. Q: Why is the induced protein insoluble (C2529)? A: T7 expression often leads to very high production of protein that can result in the target protein becoming insoluble. Potential solutions are: Induce at a lower temperature (as low as 15°C overnight) Reduce IPTG concentration to 10–40 µM Induce earlier in growth phase (OD600 = 0.3 or 0.4) and harvest cells earlier. Create an expression construct where the target protein is fused to Maltose Binding Protein (NEB #E8200). Q: Why is there no protein visible by SDS-PAGE or no activity (C2529)? A: Check for toxicity – no protein may mean the cells have lost the expression plasmid or elements of the expression plasmid have been deleted. Culture cells for protein induction. Just before induction, plate a sample on duplicate plates with and without antibiotic selection. If toxicity is an issue, there will be a significant difference between the number of colonies on the plates. Fewer colonies will be seen on plates containing antibiotic (indicating that the plasmid has been lost) compared to plates without antibiotic. If the expression plasmid is not maintained by drug selection, lysY strains will improve clone stability. Check clone integrity by restriction enzyme analysis and\/or sequencing of the ORF. Q: What are the strain properties (C2529)? A: The properties of this strain that contribute to its usefulness as a protein expression strain are described below.T7 RNA Polymerase: T7 gene1 is encoded by the lambda DE3 prophage present within the chromosome. T7 RNA polymerase is expressed from the lacUV5 promoter, which is less sensitive to catabolite repression than the wt lac promoter. Thus DE3 strains may exhibit uninduced target protein expression. Although λ DE3 is normally dormant in the host chromosome, the induction of the SOS response can occur as the result of expressing proteins that damage the E. coli chromosome, either directly or indirectly. This may lead to cell lysis. T7 Express strains do not carry the DE3 prophage and better tolerate an SOS response.Protease Deficient ([lon] ompT): E. coli B strains are \"naturally\" deficient in the Lon protease which in K-12 strains serves to degrade misfolded proteins and to prevent some cell cycle-specific proteins from accumulating. The OmpT protease resides at the surface of wild type E. coli in both K-12 and B strains, presumably helping the cells to derive amino acids from their external environment. Cells deficient in both these proteases are much more amenable to the production of proteins from cloned genes.T1 Phage Resistant (fhuA2): T1, an extremely virulent phage requires the E. coli ferric hydroxamate uptake receptor for infectivity. Deletion of this gene confers resistance to this type of phage, but does not significantly affect the transformation or growth characteristics of the cell. glmS6Ala: The glmS gene (glucosamine synthetase) is mutated and the expressed GlmS protein (67 kDa) contains six histidine to alanine substitutions (positions 62, 65, 432, 436, 466, 467). The mutated GlmS protein does not bind Ni-NTA resin in the presence of 20mM imidazole binding\/ wash buffer, whereas wt GlmS protein binds Ni-NTA resin and is not eluted until the imidazole concentration is within 55–80 mM (1).arnA::CBD The arnA gene (lipid A modification) is fused with a B. circulans ORF encoding a chitin binding domain. The expressed fusion protein is 82 kDa whereas the wt ArnA protein is 74 kDa.can::CBD The can gene (carbonic anydrase) is fused with a B. circulans ORF encoding a chitin binding domain. The expressed fusion protein is 32 kDa whereas wt carbonic anhydrase is 25 kDa.slyD::CBD The slyD gene (FKBP-type prolyl isomerase) is fused with a B. circulans ORF encoding a chitin binding domain. The expressed fusion protein is 28 kDa. However, the apparent mass of the CBD fusion is approximately 35 kDa when analyzed by SDS-PAGE. The calculated mass of wt SlyD is 21 kDa, whereas the apparent mass is 28 kDa.The GlmS(6Ala) protein and the CBD-tagged proteins are functional in vivo according to assays conducted at New England Biolabs. Q: Can I store competent cells at -20°C instead of -80°C? A: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency (TE). When tested on NEB 5-alpha Competent E.coli (NEB #C2987H), cells lost 94.5% of TE after only 24 hours of storage at -20°C. Cells lost 98.9% of TE after 2 days, and 99.6% of TE after one week of storage at -20°C. Q: Which kind of transformation tubes should be used? A: Compared to 2.0 ml tube provided with NEB single-use format competent cells, the 1.5 ml Eppendorf tube we tested worked just fine. Q: What volume of DNA can be added into competent cells? A: The volume of DNA to be added into competent cells does affect transformation efficiency. 1-5 µl of DNA (plasmid or ligation product) is recommended for 50 µl of competent cells. In 50 µl of competent cells, transformation efficiency drops to 52% when the DNA volume is increased to 10 µl (from 2 µl). Transformation efficiency drops to 18% when the DNA volume is increased to 20 µl (from 2 µl). Transformation efficiency drops to 5.2% when the DNA volume is increased to 50 µl (from 2 µl). Q: What is the shelf life for this strain (NEB #C2529H)? A: The expiration date is one year from the assay date provided with the product. Q: Are NEB's competent cells compatible with the “Mix \u0026amp; Go\" protocol? A: There is a “ Mix and Go\" protocol that provides a quick way to transform your cells by simply adding plasmid to cells and plating. No heat shock step is required. NEB has tested our competent cells in this protocol against another company's “Mix \u0026amp; Go” product. We have observed both will produce similar numbers of colonies; however, the NEB colonies are larger in size using the same incubation period. Q: How should I store SOC Outgrowth Medium? The SOC I received with my competent cells recommends storage at either room temperature or 4°C, however, when I purchase it as a stand alone product, it recommends storing it at 4°C. Which is better? A: SOC medium can be stored at either 4°C or Room Temperature depending on how fast it will be used. Storing at Room Temperature is convenient and adequate for short term usage (weeks to a couple of months). For long term storage, we recommend storing at 4°C. Please note that Outgrowth Medium 1.5 supplied with NEB #C2987R (1 x 384 well plate format) must only be stored at Room Temperature or crystals will form. Q: Is T7 expression subject to catabolite repression in BL21(DE3) and derivative strains? A: The lambda DE3 prophage carries the T7 RNA polymerase gene (T7gene1). Expression of the T7 RNA Polymerase is controlled by the lacUV5 promoter. Therefore, glucose addition will result in catabolite repression and basal protein expression from T7 promoter vectors will be better controlled when glucose is present in the media. When glycerol is the primary carbon source, there should be no effect on basal T7 expression. Q: What expression plasmids can be used with NiCo21(DE3)? A: NiCo21(DE3) is designed for protein expression from vectors which encode the phage T7 promoter. However, NiCo21(DE3) is also compatible with any promoter that is recognized by the E.coli RNA polymerase such as Plac, Ptac, Ptrc, ParaBAD, PrhaBAD and PT5-lac. The pUC19 vector is supplied as a control to test transformation efficiency. pUC19 encodes a lac promoter but due to the high-copy number we do not recommend pUC19 for protein expression. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835500515497,"sku":"C2529H","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-c2529h","provider":"Iright","version":"1.0","type":"link"}