New England Biolabs, C2987U, NEB® 5-alpha Competent E. coli (High Efficiency)

NEB 5-alpha Competent Related Categories Cloning Competent Cell Strains Applications Applications of USER® and Thermolabile USER II Enzymes,, USER, ®, Cloning,, High-throughput cloning and automation solutions, Specification Materials Required but not Supplied Nuclease-free Water (NEB #B1500) LB media LB Agar plates +/- antibiotic X-gal (optional) IPTG (optional) 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 Streptomycin 25 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: 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: How can I increase transformation efficiency? A: Addition of β-Mercaptoethanol (β-ME) to a final concentration of 24 mM has been shown to increase the transformation efficiency of NEB 5-alpha by 40%. The effect on transformation efficiency may be different when using plasmids other than pUC19. Be sure to use high purity, sterile β-ME at a stock concentration of 1.5 M. Follow the procedure below: For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes. For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice. Add 0.8 ìl of 1.5 M β-ME to 50 ìl of cells. Carefully flick the tube 4-5 times to mix cells and β-ME. Do not vortex. Incubate on ice for 10 minutes. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix. Place on ice for 5 minutes. Do not mix. Pipette 950 µl of room temperature SOC into the mixture. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. Warm selection plates to 37°C. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours. Q: How long should I incubate cells on ice after DNA has been added (NEB #C2987H and NEB #C2987I)? A: Incubating DNA with NEB 5-alpha competent cells on ice for 30 minutes is recommended. Expect approximately 20% loss in transformation efficiency when incubating for 10 minutes (see Figure on the main product page). Q: How should I calculate the transformation efficiency (C2987)? 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 µl (100 pg) of control pUC19 DNA into 50 µl of cells, outgrow by adding 250 µl of SOC and dilute 10 µl up to 1 ml in SOC before 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/.0001/.001 = 1.5 x 109 cfu/µg Q: What are the solutions/recipes (C2987)? A: SOB: 2% Vegetable peptone (or Tryptone) 0.5% Yeast Extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 SOC: SOB + 20 mM Glucose LB agar: 1% Tryptone 0.5% Yeast Extract 0.17 M NaCl 1.5% Agar Blue/White Screening: X-gal 80 µg/ml IPTG* 0.3 mM * Omit IPTG for potentially toxic genes Q: What are the strain properties (C2987)? 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 which 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: (hsdR17) 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 hsdR mutation eliminates this endonuclease activity. However, this strain has functional methyl restriction systems and may not be suitable for direct cloning of eukaryotic DNA. 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. DH5α™ is a trademark of Invitrogen Corporation. Q: What is the difference between NEB #C2987H and NEB #C2987I? A: They are the same cells with the same efficiency but provided in different formats. C2987H 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. C2987I 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 C2987I is cost effective. Refreezing the competent cells after thawing is not recommended since it will significantly reduce transformation efficiencies. Q: What is the difference between NEB #C2988J and NEB #C2987H? A: They are the same NEB 5-alpha cells just provided in different transformation efficiency and different formats. C2987H is NEB 5-alpha competent E.coli with a high efficiency of 1-3X10E9 cfu/μg pUC19 DNA. C2988J is NEB 5-alpha competent E.coli with a subcloning efficiency of >1X10E6 cfu/μg pUC19 DNA. C2987H is packaged with 20 single-use transformation tubes containing 50 μl of competent cells each. Plasmid or ligation product can be added directly into the transformation tubes for convenience. C2988J is packaged with 6 tubes containing 400 μl of competent cells each. The tubes must be thawed on ice and 50 μl of cells should be pipetted into your own transformation tubes prior to transformation. Each tube contains enough cells for 8 transformations with the benefit of reducing the cost of each transformation. Refreezing the competent cells after thawing is not recommended since it will significantly reduce transformation efficiencies. Q: What is the optimal heat shock time for this strain (NEB #C2987H and NEB #C2987I)? A: Heat shock at 42ºC for 30 seconds results in the highest transformation efficiency for NEB 5-alpha competent E.coli (NEB #C2987H and NEB #C2987I). Expect approximately 60% loss in transformation efficiency when heat shocking for 80 seconds (see Figure on the main product page). Q: What type of competent cells are suitable for transformation of DNA constructs created using Gibson Assembly? 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 10 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. 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: 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 is the shelf life for this strain (NEB #C2987H and NEB #C2987I)? A: The expiration date is one year from the assay date provided with the product. Q: How does the transformation efficiency of the 96-well plate format (NEB #C2987P) compare to the other formats? A: The transformation efficiency (TE) for the 96-well plate format NEB 5-alpha Competent E.coli (NEB #C2987P) is as high as the two other formats, NEB #C2987H and NEB #C2987I. At 1–3 x 109 cfu/μg pUC19 DNA, transformation efficiencies are 10 times higher than the TE of any other commercially available 96-well plate format competent cells. Q: What is the optimal heat shock time for the 96-well plate format NEB 5-alpha Competent E.coli (NEB #C2987P)? A: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the 96-well plate provided, 10 seconds at 42°C is optimal. Expect approximately 40% loss in transformation efficiency when heat shocking for 30 seconds. Q: I am competing in the iGEM competition. Do you have any products that I should consider from NEB? A: NEB offers NEBuilder® HiFi DNA Assembly Mix (NEB #E2621) and Golden Gate Master Mixes (NEB #E1601 and NEB #E1602), which allow for the successful assembly of multiple DNA fragments, regardless of fragment length. In addition, NEB offers DNA ladders, high-fidelity polymerases, and competent cells. Over the years, NEB has supported many iGEM teams with donated materials and scientific guidance. NEB has created this page to help you conveniently locate request forms, information on NEB products and helpful technical resources (including tips, troubleshooting guides and educational videos). Q: What is the optimal heat shock time for the 384-well plate format NEB 5-alpha Competent E. coli (NEB #C2987R)? A: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the 384-well plate provided, 15 seconds at 42°C is optimal. Expect approximately 40% loss in transformation efficiency when heat shocking for 20 seconds. Q: What is the optimal heat shock time for the 96-tube format NEB 5-alpha Competent E. coli (NEB #C2987U)? A: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the 96-tube provided, 30 seconds at 42°C is optimal. Expect approximately 15% loss in transformation efficiency when heat shocking for 20 seconds. 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 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: 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.