New England Biolabs, C3019I, NEB® 10-beta Competent E. coli (High Efficiency)

NEB 10-beta Competent Related Categories Cloning Competent Cell Strains Applications Applications of USER® and Thermolabile USER II Enzymes,, USER, ®, Cloning,, Site Directed Mutagenesis, 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 Tetracycline 15 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 (C3019)? 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): encodes for the omega-fragment of β-gal; lacX74 deletes the β-gal gene on the chromosome. pUC19 and other cloning vectors code for the α-peptide of β-galactosidase (lacZ). The α-peptide expressed from the plasmid can combine with the omega-fragment of β-galactosidase, which is expressed by the host cell. 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. Please also view NEB TV Episode 1 to learn more about Synthetic Biology. Q: What is the difference between NEB #C3019H and NEB #C3019I? A: They are the same cells with the same efficiency but provided in different formats. C3019H is packaged with 20 single-use transformation tubes, each containing 50 μl of competent cells. Plasmid or ligation product can be added directly into the transformation tubes for convenience. C3019I is packaged with 6 tubes, each containing 200 μl of competent cells. The tubes should be thawed on ice and 50 μl of cells transferred into new tubes prior to transformation. Each tube contains enough cells for 4 transformations with the benefit of reducing the cost of each transformation. If you perform 3 or 4 transformations at a time, using C3019I is cost effective. Refreezing the competent cells after thawing is not recommended since it will significantly reduce transformation efficiencies. Q: What is the shelf life for this strain (NEB #C3019H and NEB #C3019I)? 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: Does plasmid size affect transformation efficiency (C3019)? A: Yes. We transformed a series of plasmids ranging in size from 2.7 kb (pUC19) to 24 kb into NEB 5-alpha competent E.coli (NEB #C2987H) and NEB 10-beta competent E.coli (NEB #C3019H) side by side. We noted that the transformation efficiency was affected by the plasmid size. NEB 10-beta chemically competent cells are more efficiently transformed with large plasmids than NEB 5-alpha cells (see Figure on the NEB #C3019H product page). NEB 10-beta competent E.coli are recommended for the transformation of large plasmids and large assembly products generated by NEBuilder® HiFi, Gibson Assembly®, and Golden Gate Assembly reactions. Q: How should I calculate the transformation efficiency (C3019)? 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: 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: What is the optimal heat shock time for this strain (NEB #C3019H and NEB #C3019I)? A: Heat shock at 42ºC for 20-30 seconds results in the highest transformation efficiency for NEB 10-beta competent E.coli (NEB #C3019H and NEB #C3019I). Expect approximately 40% loss in transformation efficiency when heat shocking for 80 seconds (see Figure on the main product page). Q: How long should I incubate cells on ice after DNA has been added (NEB #C3019H and NEB #C3019I)? A: Incubating DNA with NEB 10-beta competent cells on ice for 30 minutes is recommended. Expect approximately 50% loss in transformation efficiency when incubating for 10 minutes (see Figure on the main product page). 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: Are NEB's competent cells compatible with the “Mix & 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 & 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: 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: Why did Synthetic Biologist Chris Voigt of MIT choose NEB 10-beta for DNA assembly and cloning? A: Can a living cell perform computation? By encoding circuits in DNA and transforming them into a living bacterial host, organisms can be manipulated to process environmental cues and have altered biological function. This repurposing of biological function is the cornerstone of synthetic biology, and is transforming markets such as biofuels and the food industry by engineering novel organisms designed for manufacture or biological sensing. Accomplishing this requires reliable assembly of large DNA constructs with specific arrangements of DNA parts, and a well-characterized organism in which to put them. The design of large DNA constructs is complex and assembly can be a formidable challenge. Chris Voigt, a synthetic biologist at the Broad Institute, and his team developed a computational environment (Cello) to standardize design of these assemblies based on user-supplied parameters and constraint. As Voigt and his team point out, the behavior of genetic circuits is dependent on many parts whose function can vary depending on genetic context, strain and growth conditions. When it came to selecting a competent cell strain, they chose NEB 10-beta Competent E. coli. Why was NEB 10-beta was chosen for Cello? Reduced recombination from RecA1 mutation Ability to clone and propagate large plasmids High transformation efficiency Better growth on M9 + glucose than some other strains Resistant to phage T1 Defined genotype Already being used in many labs around the world 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: 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. Q: Can the 96-well plate format of NEB 10-beta Competent E.coli, NEB #C3019P, be separated into smaller sections? A: Yes. For the 96-well plate format, NEB 10-beta Competent E.coli cells are packed in a 96-well divisible polypropylene plate and sealed with aluminum foil film. The plate can be snapped into four separate 24-well segments when running smaller-scale transformations. To separate a 24-well segment, place the 96-well plate on dry ice, bend the plate at the connections, and cut the foil seal between the sections. The unused sections should be returned to -80°C freezer for storage. Q: How does the transformation efficiency of the 96-well plate format (NEB #C3019P) compare to the other formats? A: The transformation efficiency (TE) for the 96-well plate format NEB 10-beta Competent E.coli (NEB #C3019P) is lower than the two other formats, NEB #C3019H and NEB #C3019I. TE of C3019P is 1-3 x 108 cfu/ μg pUC19.