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TransforMaxTM

Competent Cell

TransforMax™ EPI300™ Chemically Competent E. coli

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. 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 thetrfA gene.
  • High transformation efficiency with clones of all sizes.
  • lacZΔM15for 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.

i-transformax_bacclones.gif
 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 Chemically Competent E. coli
  • Transformation efficiency of >5 x 108 cfu/µg of pUC19.

*Covered by issued and/or pending patents.



TransforMax™ EC100™ Chemically Competent E. coli

Applications
  • Routine cloning of DNA up to 200 kb.
The highly versatile TransforMax™ EC100™ E. coli competent cells are ideal for most cloning applications. The cells provide very high transformation efficiency when tested against a wide range of supercoiled DNAs as well as DNA directly from a ligation reaction (Table 1).

Benefits
  • High transformation efficiency with clones of all sizes, including BAC clones (Table 1).
  • lacZΔM15for blue/white screening of recombinants.
  • Restriction minus [mcrA, Δ(mrr-hsdRMS-mcrBC)] enables efficient cloning of methylated DNA.
  • Endonuclease minus (endA1) to ensure high yields of DNA.
  • Recombination minus (recA1) for greater stability of large cloned inserts.

스크린샷 2013-06-25 오후 12.51.07.png
 Table 1. Comparison of the transformation efficiencies of TransforMax™ EC100™ E. coli with a variety of DNAs.Transformations were performed using 50 µl of competent cells and either supercoiled DNAs of the indicated sizes or a 1-µl aliquot from a standard 10-µl ligation reaction. Results shown are in cfu/µg of DNA and are the average transformation efficiencies obtained from several trials.


Genotype
F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) nupG



TransforMax EC100 Chemically Competent E. coli
  • Transformation efficiency of >5 x 108 cfu/µg of pUC19.



TransforMax™ EPI300™-T1R Electrocompetent E. coli


Applications
  • Construction of inducible-copy-number genomic libraries using the CopyControl™ Cloning System, with clones that are resistant to contaminating phage T1 and T5.
Phage T1-Resistant TransforMax™ EPI300™-T1R E. coli have all the benefits of the highly versatile TransforMax EPI300™E. coli competent cells with the addition of being resistant to bacteriophages T1 and T5 (tonA genotype). Once introduced into the lab environment, bacteriophage T1 rapidly lyses E. coli strains that are commonly used in cloning applications and results in significant lab downtime and the loss of valuable clones. Bacteriophage T1 is particularly difficult to eliminate from the lab and can lay dormant for many years. The tonA genotype protects the phage T1-resistant TransforMax EC100-T1Rcells and valuable clones from attack by bacteriophage T1.

Like the standard TransforMax EPI300 E. coli, this strain has been specially engineered for use with Epicentre's CopyControl™ Cloning Systems.* The cells contain an inducible mutant trfA gene whose gene product is required for initiation of replication from the oriV contained on the CopyControl pCC1™ Vectors or on clones that have been retrofitted with CopyControl capability using the EZ-Tn5™<oriV/KAN-2> Insertion Kit. This process allows TransforMax EPI300-T1R E. coli to maintain CopyControl vectors at single copy until induction occurs immediately before DNA purification.

Benefits
  • tonAfor resistance to bacteriophages T1 and T5.
  • trfAgene under tight control of an inducible promoter for copy-number control of CopyControl clones and clones retrofitted with the EZ-Tn5<oriV/KAN-2> Transposon.
  • High transformation efficiency of both large and small clones.
  • lacZΔM15for blue/white screening of recombinants.
  • Readily accepts large DNAs for construction of large-insert genomic libraries.
  • Restriction minus [mcrA, Δ(mrr-hsdRMS-mcrBC)] enables efficient cloning of methylated DNA.
  • Endonuclease minus (endA1) to ensure high yields of DNA.
  • Recombination minus (recA1) for greater stability of large cloned inserts.

Genotype

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

*Covered by issued and/or pending patents.


TransforMax™ EC100™-T1R Electrocompetent E. coli

Applications
  • Routine cloning of DNA from plasmid, fosmid, and BAC vectors, with resistance to phage T1 and T5.
Phage T1-resistant TransforMax™ EC100™-T1R E. coli have all the benefits of the highly versatile TransforMax EC100™ E. coli competent cells with the addition of being resistant to bacteriophages T1 and T5 (tonA genotype).
Phage T1-resistant TransforMax EC100-T1R E. coli are ideal for most cloning applications. However, the phage T1-resistant TransforMax EC100-T1R E. coli should not be used with Epicentre's CopyControl™ Cloning Systems.

Benefits
  • tonAfor resistance to phage T1 and phage T5.
  • High transformation efficiency with clones of all sizes–including BAC clones.
  • lacZΔM15for blue/white screening of recombinants.
  • Readily accepts large DNAs for construction of large-insert genomic libraries.
  • Restriction minus [mcrA, Δ(mrr-hsdRMS-mcrBC)] enables efficient cloning of methylated DNA.
  • Endonuclease minus (endA1) to ensure high yields of DNA.
  • Recombination minus (recA1) for greater stability of large cloned inserts.

Genotype

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



TransforMax™ EC100D™ pir+ and pir-116 Electrocompetent E. coli

Applications
  • Rescue cloning of transposition mutants created using the EZ-Tn5™ ori/KAN-2> Insertion kit or Transposome™, or the HyperMu™ ori/KAN-1>Tnp Transposome.
  • Propagation of plasmids tagged with the EZ-Tn5 ori/KAN-2> Insertion kit.
The TransforMax™ EC100D™ pir+ Electrocompetent E. coli and TransforMax™ EC100D™ pir-116 Electrocompetent E. colieach constitutively express the π protein (the pir gene product) for replication of plasmids containing the R6Kγ origin of replication (R6Kγori). The cells are derived from Epicentre's TransforMax™ EC100™ Electrocompetent E. coli by P1 phage transduction with a strain containing the pir+ or pir-116 gene linked to a dihydrofolate reductase (DHFR) marker.

The TransforMax EC100D pir+ cells will maintain R6Kγori containing plasmids at approximately 15 copies per cell,1 for cloning of potentially toxic or unstable DNA sequences. The TransforMax EC100D pir-116 cells are for high-copy propagation of up to 250 rescue plasmid copies per cell.1

Benefits
  • Accepts large clones for unbiased propagation and stability of large rescue clones.
  • lacZΔM15for blue/white screening of recombinants.
  • Restriction minus [mcrA, Δ(mrr-hsdRMS-mcrBC)] enables efficient cloning of methylated DNA.
  • Endonuclease minus (endA1) to ensure high yields of DNA.
  • Recombination minus (recA1) for greater stability of large cloned inserts.

Genotypes


TransforMax EC100D pir+ Electrocompetent E. coli:
F
F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) nupG pir+(DHFR)

TransforMax EC100D pir-116 Electrocompetent E. coli:
F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) nupG pir-116(DHFR)

Transformation Efficiency
Greater than 1 x 109 colony forming units (cfu)/µg with pR6Kan control DNA.

References
  1. Metcalf, W.W. et al. (1994) Gene 138, 1
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