Product Class: Other

Cre Recombinase
NEBU recombinant 37 70 Heat

Product Introduction

Cre Recombinase is a Type I topoisomerase.

  • Excision of DNA between two loxP sites
  • Fusion of DNA molecules containing loxP sites
  • Inversion of DNA between loxP sites

 

Catalog # Size Concentration
M0298S 50 units 1000 units/ml
M0298L 250 units 1000 units/ml
M0298M 250 units 15000 units/ml

Product Information

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. 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:

Reagents Supplied

Reagents Supplied

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

  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,

Protocols, Manuals & Usage

Protocols

  1. Protocol for Cre Recombinase (M0298)

FAQs & Troubleshooting

FAQs

  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. Why aren't the product bands from my Cre Recombinase reaction mix sharp when run on an agarose gel?
  5. Can Cre Recombinase be used to move an insert between expression vectors?

Quality, Safety & Legal

Quality Assurance Statement

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Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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