New England Biolabs, C2925I, dam-/dcm- Competent E. coli

Methyltransferase deficient 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 Kanamycin 30 µg/ml Tetracycline 15 µg/ml Shipping Notes Ships on dry ice FAQ Q: Can I transform unmethylated DNA into dam-/dcm- competent E.coli (C2925)? A: Yes. pUC19 was transformed and propagated in NEB 5-alpha (pUC19-C2987) and in dam-/dcm- (pUC19-C2925). Plasmid was purified side by side and then transformed into dam-/dcm- competent E.coli (NEB #C2925H), respectively. The transformation efficiency for methylated DNA pUC19-C2987 was 4.54 X 10E6 cfu/μg pUC19. The transformation efficiency for unmethylated DNA pUC19-C2925 was 1.09 X 10E8 cfu/ μg pUC19, which was 24X higher than that of methylated DNA. Q: Does New England Biolabs offer a methyltransferase free strain of Competent E.coli? A: Yes, dam-/dcm- Competent E.coli (NEB #C2925H) are methyltransferase deficient chemically competent E.coli cells suitable for growth of plasmids free of dam and dcm methylation. Q: How long should I incubate cells on ice after DNA has been added (NEB #C2925H and NEB #C2925I)? A: Incubating DNA with dam-/dcm- competent cells on ice for 30 minutes is recommended. When tested with pUC19, an incubation time of 20 minutes did not hurt the transformation efficiency (see Figure on the main product page). Q: Is dam-/dcm- (NEB #C2925H/C2925I) resistant to kanamycin? A: NEB #C2925H/C2925I has a low but noticeable frequency of kanamycin resistant mutations. We have observed a low, variable number of spontaneous Kan resistance mutants in this strain, and also in another commercially available dam-/dcm- strains (Invitrogen INV110). In both strains, tiny colonies appear on Kan plates at a frequency ranging from 1 in 10e6 to 1 in 10e7 cells. These tiny colonies will not grow upon restreaking on a fresh Kan plate. This level of background resistance should not be a problem for routine transformation of plasmids carrying KanR. The reason for the strain-dependent appearance of incomplete KanR is not known. Q: Is the DNA yield lower using dam-/dcm- strain (C2925)? A: No. Plasmid pUC19 was transformed into dam-/dcm- competent E.coli (NEB #C2925H) and NEB 5-alpha F´ Iq competent E.coli (NEB #C2992H), and plasmid was purified side by side. No difference between these two strains for the DNA quality or yield existed. Plasmid pBIND was also transformed into dam-/dcm- competent E.coli (NEB #C2925H) and NEB 10-beta competent E.coli (NEB #C3019H), and plasmid was purified side by side. No difference between these two strains for the DNA quality or yield existed. We recommend inoculating freshly grown colonies for plasmid propagation. Q: What are the solutions/recipes (C2925)? 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 Q: What are the strain properties (C2925)? A: The properties of this strain that contribute to its usefulness as a protein subcloning strain are described below. The genotypes underlying these properties appear in parentheses. dam and dcm Methylation Deficient (dam13::Tn9 (CamR), dcm-6) Most laboratory strains of E. coli contain both Dam methylase and Dcm methylase. Dam methylase transfers a methyl group to the adenine in the sequence GATC. Dcm methylase methylates the internal cytosine residues in the sequences CCAGG and CCTGG. Several restriction endonucleases will not cleave sites with these modified bases. The damdcm strain allows growth and purification of DNA free of Dam and Dcm methylation 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: (hsdR2) Wild type E. coli K12 strains carry the EcoK Type I 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 hsdR2 mutation described above eliminates the endonuclease. Partially Methyl Restriction Deficient: (mcrA, mcrB1 ) E. coli has a system of enzymes, mcrA, mcrB and mrr which 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. This strain has a functional Mrr endonuclease and may not be suitable for direct cloning of eukaryotic DNA. T1 Phage Resistant: (fhuA31) 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. Q: What is the difference between NEB #C2925H and NEB #C2925I? A: They are the same cells with the same efficiency but provided in different formats. C2925H 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. C925I 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 C2925I 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 #C2925H and NEB #C2925I)? A: Heat shock at 42ºC for 20-30 seconds results in the highest transformation efficiency for dam-/dcm- competent E.coli (NEB #C2925H and NEB #C2925I). The transformation efficiency is not very sensitive to the changing the heat shock time within the range of 20-60 seconds. Expect approximately 20% 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: Why were the colonies at different size after transformation of dam-/dcm- competent E.coli (C2925)? A: Dam-/dcm- (NEB #C2925H/C2925I) is a mutant strain of E.coli and the dam mutation affects this strain in a number of ways including lower transformation efficiency, lower growth rate and higher mutation rate. It is normal to get different size colonies for NEB #C2925H/C2925I. This was noticed while transforming pUC19. Both size colonies were tested and they all contained pUC19. A single large colony was picked and stirred in SOB and plated on LB/Amp. Again, both large and small colonies were visible the following morning. Q: How should I calculate the transformation efficiency (C2925)? 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 950 µl of SOC before plating 100 µl. If you count 20 colonies on the plate, the TE is: Colonies = 20 µg DNA = 0.0001 Dilution = 100/1000 = 0.1 TE = 20/.0001/0.1 = 2 x 106 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: 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 #C2925H and NEB #C2925I)? 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.