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  • FAQ: What are the strain properties of NEB 5-alpha Competent E. coli (Subcloning Efficiency)?

    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 the omega-fragment of β-galactosidase (β-gal); (argF-lacZ) deletes the β-gal gene on the chromosome. pUC19 and similar plasmids code for the α-peptide of β-gal (lacZ). The α-peptide can combine with the omega-fragment of β-gal that is carried on f80 (α-complementation). When β-gal is reconstituted in this manner it can cleave 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (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.

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