CopyCutter™ EPI400™ Chemically Competent E.coli

CopyCutter™ EPI400™ Electrocompetent E.coli

CopyCutter™ Induction Solution

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Last modified: 2008-07-15 14:57:15

CopyCutter™ EPI400™ E.coli* cells were developed to significantly lower the copy number of a wide variety of common vectors so that you can more readily clone unstable DNA sequences. DNA that is unstable at high-copy number often codes for a protein that inhibits cell growth or contains AT- and GC-rich sequences or sequences with strong secondary structure (Figure 1).

The CopyCutter™ EPI400™ cell line was derived from our high-transformation efficiency phage T1-resistant TransforMax™ EC100™ E. coli strain by manipulating a gene that controls the copy number of vectors containing ColE1 or pMB1 origins of replication (e.g., pUC- and pET-type vectors). This constitutively expressed gene, pcnB (plasmid copy number), was deleted from the TransforMax™ EC100™ strain and replaced with a modified pcnB gene that is linked to an inducible promoter, creating the CopyCutter™ EPI400™ strain.

The copy number of ColE1-type vectors in the CopyCutter™ EPI400™ strain compared to the parental TransforMax™ EC100™ strain is approximately 4- to 25-fold lower, depending on the vector. Moreover, following a short incubation in the presence of the CopyCutter™ Induction Solution, you can increase the copy number of the vector to improve plasmid yields (Figure 2).

Figure 1. DNA inserts encoding toxic gene products were successfully cloned into high-copy number vectors using CopyCutter™ EPI400™ E. coli cells After sequencing, the full-length acpP clones in TransforMAX™ EC100™ cells were found to contain multiple point mutations.	Figure 2. Uninduced CopyCutter™ EPI400™ E. coli cells containing a regB clone (Lane U) are induced to higher-copy number (Lane I) using the CopyCutter™ Induction Solution. Crude extracts of plasmid DNA were prepared from cells grown in selective media and analyzed by agarose gel electrophoresis. Approximately the same number of lysed cells (based on OD600) were loaded per lane.

Genotype

F^- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ^- rpsL nupG trfA tonA pcnB4 dhfr

CopyCutter™ EPI400™ Electrocompetent E. coli

• Transformation efficiency of >1 X 10¹⁰ cfu/µg of pUC19.
• Supplied in convenient single-use 50-µl volumes.

CopyCutter™ EPI400™ Chemically Competent E. coli

• Transformation efficiency of >1 x 10⁸ cfu/µg of pUC19.
• Supplied in convenient single-use 50-µl volumes.

Last modified: 2008-05-14 16:46:46

Benefits

• Significantly lowers the copy number of ColE1-type vectors for more reliable cloning of unstable DNA.
• Following a short incubation in the presence of an inducer, raise copy number for improved plasmid yield.
• High transformation efficiency with clones of all sizes.
• lacZΔM15 for blue/white screening of recombinants.
• Restriction minus (mcrA, Δ(mrr-hsdRMS-mcrBC)) enables
• Endonuclease minus (endA1) to ensure high yields of DNA.
• Recombination minus (recA1) for greater stability of large cloned

Last modified: 2008-05-14 16:46:46

Protocols for: CopyCutter™ EPI400™ Electrocompetent E. coli

(catalogue number C400EL10 / C400CH10 / CIS40025)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46