TransforMax™ EPI300™ E. coli cells lack the tonA gene and are engineered for use with Epicentre's CopyControl™ cDNA, Gene, and PCR Cloning Kit and other CopyControl Cloning Systems* that do not require phage T1-resistant cells. The cells contain an inducible mutant trfA gene whose gene product is required for initiation of replication from the oriV origin of replication, such as that in CopyControl pCC1™ vectors. On LB chloramphenicol plates or in LB or SOC media supplemented with chloramphenicol, CopyControl clones grown in TransforMax EPI300 E. coli replicate at single-copy number from the F-factor replicon because expression of the trfA gene is repressed. Addition of CopyControl Induction Solution induces the cells to express the trfA gene product and induces their replication at high-copy number from oriV (Figure. 1). Replication from oriV results in higher yields and higher purity of cloned DNA.
Benefits
Copy-number of clones is under tight control of an inducible promoter linked to the trfA gene. High transformation efficiency with clones of all sizes. lacZΔM15 for blue/white screening of recombinants. Restriction-minus [mcrA, Δ(mrr-hsdRMS-mcrBC)] phenotype enables efficient cloning of methylated DNA. Endonuclease-minus (endA1), to ensure high yields of DNA. Recombination-minus (recA1), for greater stability of large insert clones.
Applications
Generation of inducible copy-number clones using the CopyControl™ Cloning System.
Figure 1. Copy number of CopyControl™ BAC clones can be induced 10- to 20-fold in TransforMax™ EPI300™ E. coli. The yield of BAC DNA from CopyControl BAC clones of 110-145 kb increased >14-fold following addition of CopyControl Induction Solution. U=uninduced cells; I=induced cells.
