商品编号


C400EL10



品牌


Lucigen



公司


Lucigen



公司分类


CRISPR, Large or Difficult Fragment Cloning




10 x 50 ?l




商品信息

Optimized for cloning toxic or unstable DNA

St
ABI
lize toxic inserts in common cloning and expression vectors (pUC and pET-type vectors)

Clone and maintain challenging sequences at reduced plasmid copy number, then induce to high copy number for DNA recovery

Avoid T1 and T5 phage contamination with tonA mutation

Choose chemically competent cells for general cloning or electrocompetent cells for demanding applications such as library generation




Applications

Cloning of unstable DNA sequences or those expressing toxic proteins.

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 re
ADI
ly 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 (Fig. 2).
1

The CopyCutter EPI400 cell line was derived from Epicentre's high-transformation efficiency phage T1-resistant TransforMAX? EC100?-T1
R
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 induc
IBL
e 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, a short incubation in the presence of the CopyCutter Induction Solution can increase the copy number of the vector to improve plasmid yields (Fig. 3).


Figure 1. Copy-number of ColE1-type plasmids is lowered up to 25-fold in CopyCutter? EPI400?
E. coli
cells.
Lanes C, TransforMax? EC100? cells; Lanes U and I, uninduced or induced CopyCutter EPI400 cells. DNA extracts from the same number of lysed cells (based on OD
600
) were loaded per lane.



Figure 2. 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 3. 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 A
600
) were loaded per lane.

Benefits

Maintain clones at low-copy number, then induce to higher copy number for improved plasmid yield.

High transformation efficiency with clones of all sizes.

tonA
for resistance to bacteriophages T1 and T5.

lacZΔM15
for blue/white screening of recombinants.

Restriction minus [
mcrA, Δ(mrr-hsdRMS-mcrBC)
] genotype enables efficient cloning of methylated DNA.

Endonuclease minus (
endA1
) to ensure high yields of DNA.

Recombination minus (
recA1
) for greater st
ABI
lity of large cloned inserts.

Genotype

F
-
mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ
-
rpsL (Str
R
) nupG trfA tonA pcnB4 dhfr

CopyCutter EPI400 Electrocompetent
E. coli

Transformation efficiency of >1 x 10
10
cfu/?g of pUC19.

CopyCutter EPI400 Chemically Competent
E. coli

Transformation efficiency of >1 x 10
7
cfu/?g of pUC19.

Reference

Haskins, D. (2004)
Epicentre Forum
11(
5), 6.

*Covered by issued and/or pending patents.


ORDER INFORMATION
Ten tubes each containing 50 ?L of cells (enough cells for 10 transformations), CopyCutter? Induction Solution and pUC19 control DNA. The CopyCutter? Induction Solution is provided at a 1,000X concentration and is filter sterilized.