Standard Restriction Enzyme Reactions
Each restriction enzyme has optimal reaction (assay) conditions and different conditions for long term storage. The recommended assay and storage conditions are both determined by the manufacturer to provide the user with the highest activity, best fidelity and greatest stability for each enzyme. Factors that must be considered include temperature, pH, enzyme cofactors, salt composition, ionic strength and stabilizers. Promega restriction enzyme Reaction Buffers are designed to provide the best balance of optimal activity and convenience. Promega storage buffers have been designed after accelerated and real time/real temperature stability experiments. All enzyme storage conditions are validated through our Quality Assurance re-assay program to maximize long term stability.
Setting up digests with a single restriction enzyme is relatively straightforward. However, digests using multiple enzymes that have different buffer requirements may demand the use of alternative buffers and may require adjustments in the number of units of enzyme used. Table 3.1 lists the relative activities of restriction enzymes in Promega 10X Reaction Buffers. Alternatively, use the interactive search function of this guide to identify compatible buffers. If no compatible buffer can be found a sequential reaction may be performed in which additional buffer or salt is added to the reaction before the second enzyme, or each digest may be performed sequentially using the optimal buffers. The latter option will require either a DNA precipitation or purification step after the first digest. Regardless of the type of digest performed, the addition of BSA is recommended to stabilize the enzyme and enhance activity (1) (2) .
A. Reaction Conditions
pH: Most restriction enzymes are used between pH 7.2 and pH 8.5 as measured at the temperature of incubation. pH values outside of the optimal range may lead to star activity.
Mg2+: Commercially available restriction enzymes require Mg2+ as the only cofactor. Restriction enzyme activities are relatively insensitive to the Mg2+ concentration; similar rates are observed from 5-30mM. The presence of other divalent metal ions, especially Mn2+, may lead to star activity.
Salt Concentration: Restriction enzymes are diverse in their response to ionic strength. Most are stimulated by 50-150mM NaCl or KCl while others are inhibited by salt concentrations higher than 20mM. A few enzymes prefer acetate to chloride anions. Suboptimal ionic strength or type of ion may lead to star activity.
BSA: Bovine Serum Albumin is used in restriction enzyme storage buffers and is added to digestion reactions to stabilize the enzyme. BSA can protect restriction enzymes from proteases, non- specific adsorption and harmful environmental factors such as heat, surface tension and interfering substances. Typically, the addition of 0.1mg/ml BSA will result in a 1.5 to 6-fold enhancement of enzyme activity. The Acetylated BSA provided with Promega's restriction enzymes has been modified and extensively tested to ensure that no degrading activities are present.
Glycerol: Glycerol is added to restriction enzyme storage buffers to prevent freezing at -20°C. Repeated freeze/thawing of restriction enzymes can reduce their activity. Some restriction enzymes show reduced specificity, or increased star activity, when the glycerol concentration in the final reaction is higher than 5% although many have normal specificity at glycerol concentrations as high as 10%.
Incubation Temperature: Most restriction enzymes show maximum activity at 37°C. A few enzymes require higher or lower temperatures for optimal activity (e.g., Taq I, 65°C; Sma I, 25°C). For incubations greater than 1 hour with high temperature enzymes, cover the reactions with a drop of mineral oil to prevent evaporation. Generally, the incubation temperature for the enzyme reflects the growth temperature of the bacterial strain from which it is derived. For enzymes that have temperature optima other than 37°C, Promega provides information on percent activity at 37°C on the Product Information sheet that is packaged with each enzyme. This type of information is particularly useful when performing double digests.
Volume: Viscous DNA solutions inhibit enzyme diffusion and can reduce enzyme activity. DNA concentrations that are too dilute can fall below the Km of the restriction enzyme and also affect enzyme activity. Volume considerations must take into account final ionic strength and must result in glycerol concentrations no higher than 5-10% in order to avoid star activity. Reaction volumes of 10-50µl per microgram of DNA are recommended.