New England Biolabs, C3040I, NEB® Stable Competent E. coli (High Efficiency)

Chemically competent Related Categories Cloning Competent Cell Strains Applications High-throughput cloning and automation solutions,, Transformation Specification Antibiotic for Plasmid Selection Antibiotics for Plasmid Selection Working Concentration Ampicillin 100 µg/ml Carbenicillin 100 µg/ml Chloramphenicol 33 µg/ml Kanamycin 30 µg/ml Shipping Notes Ships on dry ice FAQ Q: Which competent cell strains are compatible with Gateway® Cloning? A: NEB 5-alpha (NEB #C2987) and NEB 10-beta (NEB #C3019) Competent E. coli can be used with Gateway Cloning. Q: What are the strain properties (C3040)? A: The properties of this strain that contribute to its usefulness as a cloning strain are described below. The genotypes underlying these properties appear in parentheses. Blue/White Screening (Φ80 Δ(lacZ)M15): makes omega-fragment of β-gal; lacX74 deletes the β-gal gene on the chromosome. pUC19 and similar plasmids code for the α-peptide of β-galactosidase (lacZ). The α-peptide can combine with the omega-fragment of β-galactosidase that is carried on the F' (α-complementation). When β-galactosidase is reconstituted in this manner it can cleave X-gal and results in blue colonies on an X-gal plate. Inserts cloned into the plasmid polylinker disrupt the α-peptide gene and the colonies are white. Recombination Deficient: (recA1) E. coli has a repair system that will recombine homologous sequences. Genomic clones often have duplicated regions, and recA mediated rearrangements can be problematic, particularly when regions of homology are longer than 50 bp. Strains that have the recA function deleted tend to grow more slowly than recA+ strains. Endonuclease I Deficient (endA1): The periplasmic space of wild type E. coli cells contains a nonspecific endonuclease. Extreme care must be taken to avoid degradation of plasmids prepared from these cells. The endA mutation deletes this endonuclease and can significantly improve the quality of plasmid preparations. Restriction Deficient [Δ(mrr-hsdRMS-mcrBC)]: Wild type E. coli K12 strains carry a restriction endonuclease which cleaves DNA with sites (AAC(N6)GTGC and GCAC(N6)GTT. While E. coli DNA is protected from degradation by a cognate methyl-transferase, foreign DNA will be cut at these sites. The deletion described above eliminates both the methylase and the endonuclease. Methyl Restriction Deficient [mcrA Δ(mrr-hsdRMS-mcrBC)]: E. coli has a system of enzymes, mcrA, mcrB and mrr that will cleave DNA with methylation patterns found in higher eukaryotes, as well as some plant and bacterial strains. DNA derived from PCR fragments, cDNA or DNA previously propagated in E. coli will not be methylated at these sites and will not be cleaved. All three Mcr enzymes have been inactivated in NEB 10-beta allowing the introduction of eukaryotic DNA of genomic origin (e.g. primary libraries) if desired. T1 Phage Resistant (fhuA): 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. Lac Promoter Control: (lacIq) The lac repressor blocks expression from lac, tac and trc promoters frequently carried by expression plasmids. If the level of lac repressor in E. coli cells is not sufficient to inhibit expression via these promoters during transformation or cell growth, even low levels of expression can reduce transformation efficiency and select against desired transformants. The extra molecules of lac repressor in lacIq strains help to minimize promoter activity until IPTG is added. Q: What is the shelf-life for this strain? A: The expiration date is one year from the assay date provided with the product. Q: Which strain of Competent E.coli should I use for general cloning? A: NEB 5-alpha Competent E. coli (NEB #C2987) is a high efficiency derivative of DH5α™, the industry standard cloning strain. NEB Turbo Competent E. coli (NEB #C2984) and NEB 5-alpha F´Iq Competent E. coli (NEB #C2992) allow potentially toxic genes to be cloned due to tight control of expression by lacIq and are suitable for blue/white screening. NEB Turbo Competent E. coli (NEB #C2984) brings unmatchable speed to your transformations with visible colonies after just 6.5 hours and plasmid preparation capability after 4 hours. NEB 10-beta Competent E. coli (NEB #C3019) is a derivative of DH10B™ and can be used for transforming large plasmids and BACs. Dam-/dcm- Competent E. coli (NEB #C2925) can be used for methylation-free plasmid growth. If cloning efficiency is negatively affected by repetitive DNA elements in the vector or insert sequence, then NEB Stable Competent E. coli (NEB #C3040) is recommended. (See Protocol for cloning DNA containing repeat elements). Not sure which cloning strain to choose? The NEB Cloning Competent E.coli Sampler (NEB #C1010) allows you to sample 4 of our popular chemically competent strains. Q: Are NEB Stable Competent E. coli cells suitable for transformation of large plasmids and large NEBuilder HiFi, Gibson Assembly or Golden Gate Assembly products? A: Yes, chemically competent NEB Stable is recommended for large plasmids and large NEBuilder HiFi, Gibson and Golden Gate Assembly products. Q: How should I calculate the transformation efficiency (C3040)? A: Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 µg of plasmid into a given volume of competent cells. The term is somewhat misleading in that 1 µg of plasmid is rarely actually transformed. Instead efficiency is routinely calculated by transforming 100 pg-1 ng of highly purified supercoiled plasmid under ideal conditions. If you plan to calculate efficiency to compare cells or ligations, keep in mind the many variables which affect this metric. Transformation efficiency (TE) equation: TE = Colonies/µg/Dilution Colonies = the number of colonies counted on the plate µg = the amount of DNA transformed expressed in µg Dilution = the total dilution of the DNA before plating TE calculation example: Transform 2 ul (100 pg) of control pUC19 DNA into 50 ul of cells, outgrow by adding 250ul of NEB 10-beta/Stable Outgrowth Medium and dilute 10 ul up to 1 ml in NEB 10-beta/Stable Outgrowth Medium prior to plating 30 µl. If you count 150 colonies on the plate, the TE is: Colonies = 150 µg DNA = 0.0001 Dilution = 10/300 x 30/1000 = 0.001 TE = 150/0.0001/0.001 = 1.5 x 109 cfu/µg Q: What is the optimal heat shock for this strain? (C3040) A: Heat shock at 42ºC for 30 seconds results in the highest transformation efficiency for NEB Stable Competent E.coli. 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: Is NEB Stable suitable for larger plasmid transformations? A: Yes. NEB has successfully transformed a 24 kb plasmid. When an equal amount of DNA molecules were transformed, the transformation efficiency for the 24 kb plasmid was approximately 80% relative to pUC19 (which is 2.7 kb in size). Q: Is NEB Stable a substitute for Stbl2 or Stbl3? A: Yes, NEB Stable is recommended in all difficult cloning experiments. The genotype of NEB Stable is not related to the Stbl2 or Stbl3 cell line but the performance of NEB Stable is superior. The performance of all 3 strains was tested by cloning of a DNA fragment with 5 x 32bp repeats. The test construct is named pUC-5xREP and it was created using the Gibson Assembly method of cloning. Following transformation of the Gibson Assembly reaction into the respective strain, 5xREP insert stability was analyzed by colony PCR: Stbl2 colonies achieved 1-mm diameter in approximately 24 hrs at 30°C. This population of colonies all carried plasmid clones with deletions (33 of 33 colonies analyzed). Stbl3 colonies achieved 1-mm diameter in 18-20 hrs at 30°C. Of 33 colonies analyzed, 22 contained full-length 5xREP insert (67% correct). NEB Stable cells grow well at 30°C and 1-mm diameter colonies are obtained after 18-20 hrs at 30°C. 5xREP insert stability was best when using NEB Stable as the cloning strain (97% correct, 32 of 33 colonies analyzed). Importantly, NEB Stable carries the endA mutation (in contrast to Stbl3) so that isolated plasmids are free of Endonuclease I. Q: What type of competent cells are suitable for transformation of DNA constructs created using NEBuilder HiFi DNA Assembly Master Mix? A: The resulting DNA constructs are compatible with most E. coli competent cells. NEB recommends using NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987). If the assembled products are larger than 15 kb, NEB recommends using NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019) or NEB 10-beta Electrocompetent E. coli (NEB #C3020). If the assembled genes contain repetitive sequences, NEB Stable Competent E. coli (NEB #C3040) should be used. Not sure which cloning strain to choose? The NEB Cloning Competent E.coli Sampler (NEB #C1010) allows you to sample 4 of our popular chemically competent strains. Q: How should I store the NEB 10-beta/Stable Outgrowth Medium? A: The NEB 10-beta/Stable Outgrowth 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. Q: Do NEB Stable Competent E.coli require an outgrowth recovery period after transformation with AmpR plasmids? A: Transformation reactions with AmpR plasmids do not require an outgrowth period after addition of Outgrowth Medium. Plasmid selection using antibiotics other than ampicillin requires an outgrowth period of 60 minutes at 30°C before plating on selective media. 30°C or 37°C may be used for plate incubation, however 30°C is recommended as some constructs may be unstable at elevated temperatures. Q: How should fragments be prepared for assembly using NEBuilder HiFi? A: Fragments can be prepared by the following methods: PCR-generated fragments can be cleaned-up by using Monarch PCR column or Exo-CIP Rapid PCR Cleanup Kit if amplicon purity is greater than 95%. If plasmid DNA was used as template during PCR, it can be removed by DpnI treatment if necessary. If multi-bands are observed, we recommend optimizing the PCR. If this is not possible gel purification is recommended. Gel extraction can introduce guanidine thiocynate (from the dissolving buffer) that can reduce the efficiency of the assembly reaction. To minimize this contamination, trim the gel slice so that a smaller amount of gel dissolving buffer can be used. Restriction enzyme digestion of a plasmid can be performed followed by heat-inactivation or column purification. Commercially ordered fragments can be re-suspended in nuclease-free water or TE buffer and directly used in the assembly reaction.