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Applications
- Generation of inducible copy-number clones using the CopyControl™ Cloning
System.
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 or
in clones that are retrofitted with CopyControl capability using the EZ-Tn5™<oriV/KAN-2> Insertion
Kit. 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 (Fig. 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.
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| 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. |
Genotype
F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74
recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) nupG trfA dhfr
TransforMax EPI300 Electrocompetent E. coli
- Transformation efficiency of >1 x 1010 cfu/µg of pUC19.
TransforMax EPI300 Chemically Competent E. coli
- Transformation efficiency of >5 x 108 cfu/µg of pUC19.
*Covered by issued and/or pending patents.
Colony Fast-Screen™ Kit (PCR Screen)
Colony Fast-Screen™ Kit (Restriction Screen)
Colony Fast-Screen™ Kit (Size Screen)
CopyControl™ BAC Cloning Kits (BamH I, EcoR I, or Hind III) CopyControl™ pCC1BAC™ Cloning-Ready Vectors (BamH I, EcoR I, or Hind III)
CopyControl™ cDNA, Gene and PCR Cloning Kits
CopyControl™ Fosmid Library Production Kit CopyControl™ HTP Fosmid Library Production Kit
Electroporation Cuvettes
EZ-Tn5™ <oriV/KAN-2> Insertion Kit
TransforMax™ EPI300™-T1R Electrocompetent E. coli TransforMax™ EPI300™-T1R Chemically Competent E. coli
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