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 the 1 kb Plus DNA Ladder (NEB# N0469).
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).
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
M0298S
-20
Cre Recombinase
M0298SVIAL
-20
1 x 0.05 ml
1,000 units/ml
Cre Recombinase Reaction Buffer
B0298SVIAL
-20
1 x 1.5 ml
10 X
Control DNA Linearized pLox2+
N0416SVIAL
-20
1 x 0.016 ml
0.125 mg/ml
M0298L
-20
Cre Recombinase
M0298LVIAL
-20
1 x 0.25 ml
1,000 units/ml
Cre Recombinase Reaction Buffer
B0298SVIAL
-20
1 x 1.5 ml
10 X
Control DNA Linearized pLox2+
N0416SVIAL
-20
1 x 0.016 ml
0.125 mg/ml
M0298M
-20
Cre Recombinase
M0298MVIAL
-20
1 x 0.017 ml
15,000 units/ml
Cre Recombinase Reaction Buffer
B0298SVIAL
-20
1 x 1.5 ml
10 X
Control DNA Linearized pLox2+
N0416SVIAL
-20
1 x 0.016 ml
0.125 mg/ml
Properties & 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 Buffer
15 mM Tris-HCl
250 mM NaCl
0.3 mg/ml BSA
50% Glycerol
pH 8 @ 25°C
Heat Inactivation
70°C for 10 minutes
Product Notes
Incubation of the Cre Recombinase reaction mix at 70°C for 10 minutes is recommended before agarose gel analysis.
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.
Longer incubation times will not improve recombination, and instead, will likely lead to higher molecular weight recombination products.
Increasing the amount of Cre Recombinase in the reaction can inhibit recombination by forming loxP dependent Cre-DNA aggregates.
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
Abremski, K. and Hoess, R. (1984). J. Biol. Chem.. 259,
Abremski, K. et al. (1983). Cell. 32, 1301-1311.
Metzger, D. and Feil, R (1999). Curr. Opin. Biotechnol.. 10,
Cantor, E. and Chong, S. (2001). Protein Expr. Purif.. 22,
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