To use oligos effectively, the stoichiometry of the reaction needs to be established beforehand by performing a series of titrations and identifying the optimum range for the concentration of oligos. Addition of duplex DNA containing a recognition site can compete for binding and reduce the effective enzyme concentration. The oligos provide the additional recognition site needed to activate substrate-bound, but dormant enzyme monomers. If that is not possible, add duplex oligonucleotides that contain the recognition site.As a starting point, we recommend using 1-2 μl of restriction enzyme (at the supplied units/μl) per microgram of substrate and performing 2-fold serial dilutions of the enzyme, keeping the DNA concentration constant. Titrate the units of enzyme used in the reaction to determine the optimal enzyme to substrate ratio.If you are using an enzyme that may require more than one recognition site on the substrate to cleave optimally, we suggest two possible optimization methods: Please visit "Restriction Enzyme Cleavage: ‘single-site’ enzymes and ‘multi-site’ enzymes” for more information. These enzymes are indicated with the icon. This table indicates which NEB restriction enzymes have been identified to require binding at more than one recognition site for efficient cleavage. Restriction enzymes requiring multi-sites for efficient cleavage
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