1. Reaction Conditions
2. Single Restriction Enzyme Digests
3. Multiple Restriction Enzyme Digests
4. Experimental Controls
5. References

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's 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's 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 K_m 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.

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B. Single Restriction Enzyme Digests

An analytical restriction enzyme reaction is usually performed in a volume of approximately 20µl on 0.2-1.5µg of substrate DNA using a 2- to 10-fold excess of enzyme over DNA, based on unit definition. Use of an unusually large volume of DNA or enzyme may give aberrant results. Caution should be exercised to prevent higher than normal concentrations of EDTA and glycerol. The following is an example of a typical analytical single restriction enzyme digestion:

1. Under sterile conditions add the following components, in the order stated, to a sterile microcentrifuge tube.

Sterile, nuclease-free water	14µl
Restriction enzyme 10X buffer	2µl
BSA, Acetylated (1mg/ml)	2µl
DNA sample 0.2-1µg, in water or TE buffer	1µl
Restriction enzyme, 2-10U	1µl
Final volume	20µl

2. Mix gently by pipetting. Centrifuge briefly at 12,000 x g in a micro centrifuge to collect the contents at the bottom of the tube.

3. Incubate at the optimum temperature for 1-4 hours.

4. Add 4µl of Blue/Orange 6X Loading Dye (or another appropriate DNA loading buffer), and proceed to gel analysis.

Larger scale restriction enzyme digestions can be accomplished by scaling this basic reaction proportionately.

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C. Multiple Restriction Enzyme Digests

If all of the restriction enzymes in a multiple digest have the same optimal buffer, setting up the digest is straightforward. However, when this is not the case, several options are available.

1. Use the optimal buffer supplied with one enzyme if the activity of the second enzyme is acceptable in that same buffer. Alternatively, acceptable activity for both enzymes may be achieved by using another of Promega’s 4-CORE^® 10X Buffers (Cat.# R9921). If one of the enzymes has less than 75% activity in the chosen buffer, the reaction time or the number of units of enzyme used may need to be increased. Be aware of possible star activity under non-optimal reaction conditions (see Table 3.1 or use the interactive search function of this guide to identify compatible buffers).

2. Choose an isoschizomer or neoschizomer with more compatible buffer requirements.

3. Perform a single digest with the first enzyme then inactivate that enzyme. Add the ingredients necessary for the second digest then add the second enzyme. For example, use a lower salt buffer and enzyme first, then inactivate the first enzyme, add enough salt to achieve the concentration required for the second digest, and add the second restriction enzyme.

4. Perform each digest sequentially using the optimal buffers. This will require either a DNA precipitation or purification step after the first digest. Although this procedure involves more steps than those listed above, in situations where options 1-3 are not satisfactory, it may be the best alternative.

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D. Experimental Controls

Some common controls used for restriction enzyme digestion and gel analysis are given in Table 2.1.

Table 2.1. Restriction Enzyme Reaction Controls.

1. Perform a digestion as described in the unit definition for the enzyme but using the experimentally derived DNA instead of control DNA. Adjust the number of enzyme units based on recognition site density.
2. Perform the experimental digest, replacing the experimental DNA with the same quantity of commercial quality DNA (usually lambda DNA). Adjust the number of enzyme units based on recognition site density.

1. Determining the distance samples have run in the gel.
2. Measuring the sizes of unknown fragments.
3. Repeatedly seeing a familiar pattern of known and standardized MW.

Anomalous mobility due to differences in the quantity of sample loaded may also be detected.

1. Williams, R., Kline, M. and Smith, R. (1996) BSA and restriction enzyme digestions. Promega Notes 59, 46.
2. Lepinske, M. (1996) BSA and restriction enzyme digestions. Promega Notes 60, 28.
3. Protocols and Applications Guide. Third Edition. (1996) Promega Corporation.