1 unit is defined as the amount of enzyme required to achieve the maximum site-specific recombination of 250ng of control DNA in 30 minutes at 37℃ in a 50μl reaction system. The maximum recombination can be analyzed by agarose gel electrophoresis or by screening on the corresponding resistance plates after transformation of the reactions.
Application：
Cre Recombinase is commonly used to catalyze the site-specific recombination of sequences between loxP sites.Definition of activity:1 unit is defined as the amount of enzyme required to achieve the maximum site-specific recombination of 250ng of control DNA in 30 minutes at 37℃ in a 50μl reaction system. The maximum recombination can be analyzed by agarose gel electrophoresis or by screening on the corresponding resistance plates after transformation of the reactions.Enzyme stock solution: 15mM Tris-HCl (pH8.0), 250mM NaCl, 0.3mg/ml BSA, 50% (v/v) Glycerol.Inactivation or inhibition: Inactivation by heating at 70℃ for 10 minutes.
Source：
Recombinant Cre Recombinase expressed in E. coli
Inactivation or inhibition：
Inactivation by heating at 70℃ for 10 minutes.
Precautions：
Before examining DNA samples treated with this product by agarose gel electrophoresis, it is recommended to perform heat inactivation of the reaction mixture at 70℃ for 10 minutes.Since the reaction catalyzed by Cre Recombinase is a dynamic equilibrium process, only 20-30% of the DNA molecules undergoes recombination.Prolonging the incubation time does not improve the recombination efficiency, but may lead to the formation of high molecular weight recombination products.Excessive amount of Cre Recombinase in the reaction will form a loxP site-dependent Cre-DNA complex (the DNA band larger than 6kb in Figure 2), which may inhibit the recombination reaction.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
1.Thaw the reagents and mix well before use. Place the Cre Recombinase on ice.2.Set up the following reaction on ice.Note: When multiple reactions are required, prepare a master mix including all reagents except the substrate DNA and then dispense to different nuclease-free PCR tubes. Finally, add substrate DNA to each tube. Reagent Volume Final concentration Nuclease-freeWater 44-x - 10X Cre RecombinaseBuffer 5 1X SubstrateDNA x 5ng/μl* Cre Recombinase 1 1U/50μl Finalvolume 50 - *For reference only, the DNA amount can be adjusted depending on the experimental requirements.3.Gently mix by pipetting or vortex, and centrifuge briefly at room temperatur4.Incubate at 37℃ for 30 minutes.5.Inactivate the Cre Recombinase by incubation the reaction mixture at 70℃ for 10 minutes.6.Analyze the recombination result by agarose gel electrophoresis. In the case of plasmid recombination, the recombinant plasmid can be identified by screening single colonies after bacteria transformation.References:1.Pinkney JN, Zawadzki P, Mazuryk J, Arciszewska L K, Sherratt D J, Kapanidis A N. Proc Natl Acad Sci USA. 2012. 109(51):20871-6.2.Kühn R, Torres RM. Methods Mol Biol. 2002. 180:175-204.Related Products: Cat. No. Product Name Pack Size D2608-1μg pCMV-Cre-EGFP 1μg D2608-100μg pCMV-Cre-EGFP 100μg D2607-1μg pCMV-Cre-mCherry 1μg D2607-100μg pCMV-Cre-mCherry 100μg D0508S Genome-Editing Mutation Detection Kit 25T D0508M Genome-Editing Mutation Detection Kit 100T D7080S T7 Endonuclease I 250U D7080M T7 Endonuclease I 1250U D7080L T7 Endonuclease I 5000U
