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Order | Protocol | Citations | Related Products

Applications

• Rescue cloning of transposition mutants created using the EZ-Tn5™ <R6Kγori/KAN-2> Insertion kit or Transposome™, or the HyperMu™ <R6Kγori/KAN-1>Tnp Transposome.
• Propagation of plasmids tagged with the EZ-Tn5 <R6Kγori/KAN-2> Insertion kit.

The TransforMax™ EC100D™ pir⁺ Electrocompetent E. coli and TransforMax™ EC100D™ pir-116 Electrocompetent E. coli each 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.
• lacZΔM15 for 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^- mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (Str^R) 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 (Str^R) 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.

You may wish to consider the following related products:

Electroporation Cuvettes
EZ-Tn5™ <R6Kγori/KAN-2> Insertion Kit
EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ Kit
EZ-Tn5™ Transposon Construction Vectors
HyperMu™ <R6Kγori/KAN-1>Tnp Transposome™ Kit

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