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DNA Modifying Enzymes and Cloning >
Recombinases >
Cre Recombinase |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
- Supplied with 2 μg control DNA
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)
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).
Applications:- Excision of DNA between two loxP sites
- Fusion of DNA molecules containing loxP sites
- Inversion of DNA between loxP sites
Reagents Supplied:
pLox2+ (linearized)
Cre Recombinase Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
40 units at 70°C for 10 minutes
Specific Activity:
11,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X Cre Recombinase Reaction Buffer
Incubate at
37°C.
1X Cre Recombinase Reaction Buffer:
50 mM Tris-HCl
33 mM NaCl
10 mM MgCl2
pH 7.5 @ 25°C
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.
Concentration:
1,000 units/ml
Storage Conditions:
15 mM Tris-HCl
250 mM NaCl
50% Glycerol
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- Control DNA: Linearized pLox2+ is 3.65 kb in length, with a loxP site approximately 350 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, ampicillin-resistant plasmid (which migrates at approximately 1.7 kb on a 0.8% agarose gel) and one 850 bp DNA fragment.
Usage 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.
FAQs
- What is the protein concentration of NEB's Cre Recombinase?
- What is the molecular weight of Cre Recombinase?
- Can Cre Recombinase be used to linearize a BAC containing a loxP site?
- What is the sequence of the loxP sites in the pLox2+ control DNA to be used with Cre Recombinase?
- Is the Cre Recombinase control DNA available as a separate product?
- Why aren't the product bands from my Cre Recombinase reaction mix sharp when run on an agarose gel?
- Can Cre Recombinase be used to move an insert between expression vectors?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of ΦX174 RF I DNA (HaeIII digest)
and 5 units of Cre Recombinase incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 5 units of Cre Recombinase with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
References
- Abremski, K. and Hoess, R. (1984) J. Biol. Chem., 259, 1509-1514.
- Abremski, K. et al. (1983) Cell, 32, 1301-1311.
- Metzger, D. and Feil, R. (1999) Curr. Opin. Biotechnol., 10, 470-476.
- Cantor, E. and Chong, S. (2001) Protein Expr. Purif., 22, 135-140.
Reagents Sold Separately
pLox2+ (linearized)
Cre Recombinase Reaction Buffer
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