New England Biolabs, C2992H, NEB® 5-alpha F'Iq Competent E. coli (High Efficiency)

Having trouble opening your tubes? 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 Streptomycin 25 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: Does plasmid size affect transformation efficiency (C2992)? A: Yes. We transformed a series of plasmids ranging in size from 2.7 kb (pUC19) to 24 kb into NEB 5-alpha F´Iq competent E.coli (NEB #C2992H) 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 F´Iq cells. Q: How long should I incubate cells on ice after DNA has been added (NEB #C2992H and NEB #C2992I)? A: Incubating DNA with NEB 5-alpha F´Iq competent cells on ice for 30 minutes is recommended. When tested with pUC19, an incubation time of 10 minutes did not significantly lower the transformation efficiency (see Figure on the main product page). Q: How should I calculate the transformation efficiency (C2992)? 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 = 150/0.0001/0.001 = 1.5 x 109 cfu/µg Q: What are the solutions/recipes (C2992)? 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 (C2992)? 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: (F' Δ(lacZ)M15)makes omega-fragment of β-gal; Δ(lac-proAB) 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 which 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: (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. M13 phage sensitive: (F´) Infection by M13 and other similar phage requires E. coli surface features conferred by the F plasmid carried by some E. coli strains. Infection by these phage allows production of single-stranded DNA and the generation of phage display libraries. The F plasmid is frequently modified to carry other useful DNA in the cell [e.g. Δ(lacZ)M15 in this cell line] and when modified is called F´. 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. 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.DH5α™ is a trademark of Invitrogen Corporation. Q: What is the difference between NEB #C2992H and NEB #C2992I? A: They are the same cells with the same efficiency but provided in different formats. C2992H 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. C2992I 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 C2992I is cost effective. 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 #C2992H and NEB #C2992I)? A: Heat shock at 42ºC for 30 seconds results in the highest transformation efficiency for NEB 5-alpha F´Iq competent E.coli (NEB #C2992H and NEB #C2992I). Expect approximately 70% loss in transformation efficiency when heat shocking for 80 seconds (see Figure on the main product page). 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: 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 #C2992H and NEB #C2992I)? 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 & 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: 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.