New England Biolabs, C3020K, NEB® 10-beta Electrocompetent E. coli

Supplied with outgrowth medium optimized for NEB 10-beta & NEB Stable Competent Related Categories Cloning Competent Cell Strains Applications 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 Tetracycline 15 µg/ml Shipping Notes Ships on dry ice FAQ Q: How should I calculate the Electrotransformation efficiency (C3020)? 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 electrocompetent cells. For example, if a 10 pg transformation with pUC19 yields 100 colonies when 100 µl of a 1:100 dilution is plated, then the cfu/µg is 1.0 x 1010 (100 cfu/10pg x 106 pg/µg x 1 ml/0.1 ml plated x 100 dilution). Q: What are the strain properties (C3020)? 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: (recA) E. coli has a repair system which 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 are recA- tend to grow more slowly than recA+ strains. Endonuclease I Deficient: (endA) 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 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. 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. 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: 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 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.