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  • Cre Recombinase

    Description

    Cre Recombinase is a Type I topoisomerase from bacteriophage P1 that catalyzes the site-specific recombination of DNA between loxP sites (1). The enzyme requires no energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction products (2). The loxP recognition element is a 34 base pair (bp) sequence comprised of two 13 bp inverted repeats flanking an 8 bp spacer region which confers directionality (3). Recombination products depend on the location and relative orientation of the loxP sites. Two DNA species containing single loxP sites will be fused. DNA between directly repeated loxP sites will be excised in circular form while DNA between opposing loxP sites will be inverted with respect to external sequences.

    Figure 1. Cre Recombinase Reaction with loxP 2+ control substrate. The reactions yields a 20-30 % recombination. Marker M is 2-Log DNA Ladder (NEB# N0469).

    Highlights

    • Isolated from a recombinant source
    • Excision of DNA between two loxP sites
    • Fusion of DNA molecules containing loxP sites
    • Inversion of DNA between loxP sites

    Product Source

    Purified from an E. coli strain carrying a plasmid encoding Cre Recombinase from bacteriophage P1 with additional N-terminal Ala and Gly residues (4).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Cre Recombinase Reaction Buffer-2010X
    Control DNA Linearized pLox2+125 ng/μl

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme necessary to produce maximal site-specific recombination of 0.25 μg pLox2+ control DNA in 30 minutes at 37°C in a total reaction volume of 50 μl. Maximal recombination is determined by agarose gel analysis and by transformation of reactions followed by selection on ampicillin plates.

    Reaction Conditions

    1X Cre Recombinase Reaction Buffer
    Incubate at 37°C

    1X Cre Recombinase Reaction Buffer:
    33 mM NaCl
    50 mM Tris-HCl
    10 mM MgCl2
    pH 7.5 @ 25°C

    Usage Concentration

    1,000 - 15,000 units/ml

    Storage Temperature

    -20°C

    Storage Conditions

    15 mM Tris-HCl
    250 mM NaCl
    0.3 mg/ml BSA
    50% Glycerol
    pH 8.0 @ 25°C

    Heat Inactivation

    70°C for 10 min

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

    Notes

    1. Incubation of the Cre Recombinase reaction mix at 70°C for 10 minutes is recommended before agarose gel analysis.
    2. Because the Cre Recombinase reaction is an equilibrium reaction, we observe 20-30% recombination on our loxP 2+ control substrate (Fig. 1). This modest yield produces a faint band on an ethidium bromide stained gel and the concomitant reduction in substrate staining intensity.
    3. Longer incubation times will not improve recombination, and instead, will likely lead to higher molecular weight recombination products.
    4. Increasing the amount of Cre Recombinase in the reaction can inhibit recombination by forming loxP dependent Cre-DNA aggregates.
    5. Control DNA: Linearized pLox2+ is 3,625 bp in length, with a loxP site approximately 400 bp from each end. Between the loxP sites lie an origin of replication and ampicillin-resistance gene. Recombination between these loxP sites produces a circular (2,787 bp), ampicillin-resistant plasmid (which migrates at approximately 1.7 kb on a 0.8% agarose gel) and one 838 bp DNA fragment.

    References

    1. Abremski, K. and Hoess, R. (1984). J. Biol. Chem.. 259
    2. Abremski, K. et al. (1983). Cell. 32, 1301-1311.
    3. Metzger, D. and Feil, R (1999). Curr. Opin. Biotechnol.. 10
    4. Cantor, E. and Chong, S. (2001). Protein Expr. Purif.. 22

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What is the molecular weight of Cre Recombinase?
    2. Can Cre Recombinase be used to linearize a BAC containing a loxP site?
    3. What is the sequence of the loxP sites in the pLox2+ control DNA to be used with Cre Recombinase?
    4. Is the Cre Recombinase control DNA available as a separate product?
    5. Why aren't the product bands from my Cre Recombinase reaction mix sharp when run on an agarose gel?
    6. Can Cre Recombinase be used to move an insert between expression vectors?
    1. Protocol for Cre Recombinase (M0298)