Home  Careers  Contact Us About Viswagen Products Ordering Information Restriction Endonuclease Standard Quality Control Unit Definition One unit of restriction endonuclease activity is defined as the amount of enzyme required to produce a complete digest of 1 µg of substrate DNA (or fragments) in a total reaction volume of 50µl in 60 minutes under optimal assay conditions as stated for each restriction endonucease. Determination of the volume activity of restriction endonucleases Restriction endonuclease activity assays are performed by adding different enzyme dilutions to the appropriate assay buffer containing 1 µg of substrate DNA. After 60 minutes incubation at the appropriate temperature, the digestion is stopped and the DNA samples are visualized by agarose gel electrophoresis. The most diluted enzyme solution giving a complete digest is used to calculate the activity in units/µl. Please note that the activity is substrate dependent and when working with a new substrate, the enzyme should be titrated to determine the actual or expected activity. Quality Controls The results of all quality contol assays are reported on the Technical Data Sheet provided with each enzyme. Overdigestion Assay SBS uses an overdigstion assay as a qualitative determination of enzyme purity and of a lack of nonspecific DNases. In the overdigestion assay, increasing amounts of each restriction endonuclease (usually10, 20, 30, 40, 50units) are added to a series of tubes containing 1µg substrate DNA. After a 20-hours incubation under the recommended assay conditions, the maximum number of units giving a clear, sharp, normal banding pattern is determined by agarose gel electrophoresis. To pass the test, the enzyme must yield an unaltered banding pattern under conditions of up to 800-fold overdigestion (units×hours) as compared to a 2-fold digest. If enzyme exhibits "star" activity at a lower than 800-fold functional excess, the product description includes information on the functional excess at which the "star" activity does not occur. Assay for Non-Specific Endonucleases To assay for nonspecific endonuclease contamination, each restriction endonuclease is incubated with a supercoiled plasmid substrate lacking the recognition sequence of the restriction endonuclease. A single nonspecific nick in the RF I DNA converts it to the RF II form (nicked circle). Increasing amounts of enzyme (usually 10, 20, 30, 40, 50units) are added to a series of tubes containing 1µg of RF I (supercoiled form) DNA. After a 20-hours incubation under the recommened assay conditions, the two forms are distinguished on gels and the percent conversion from RF I to RF II is determined. Ligation and Recutting Assay SBS uses a ligation assay to determine the functional purity of the DNA after restriction enzyme digestion. Substrate DNA is completely digested with a 10- and 50-fold excess of the restriction endonuclease in the appropriate assay buffer, ligated with T4 DNA Ligase and recut with the same restriction enzyme. Cut, ligated and recut DNAs are analyzed by agarose gel electrophoresis. A normal banding pattern indicates intact 5' and 3' temini as well as the absence of contaminating nucleases or phosphatases. Stability All SBS restriction endonuclease are reassayed every 4-6 months. This process allows us to ensure full enzyme activity and optimal performance in every enzyme we ship. Due to the excellent results of this testing, we have extended the expiration dates of most of our enzyme to 18 mouths. The corresponding Commercially Available Isoschizomers According to the nomenclature of restriction endonucleases, newly discovered restriction enzymes possessing a unique specificity are called prototypes. Restriction endonucleases with the specificity of a prototype, delineated later, are called isoschizomers. Fifteen isoschizomers sold at SBS have names which might be not familiar to you. The table below allow you to find the enzyme of interest easily. Notes Please keep in mind that different isoschizomers with the same specificity, supplied by different companies, could be of distinct origin and may vary in optimal reaction conditions or other properties. For this reason we recommend strongly the use of original SBS assay conditions to achieve DNA cleavage. Information on commercially available restriction endonucleases is from: Roberts, R.J., Restriction Enzyme Database Copyright ©.,NEB Inc., REBASE 2000. Restriction Enzyme Buffer Guide According to assay conditions SBS has divided the restriction endonucleases into five groups. Each group is more active in one of the five following reaction buffers: Low salt buffer 10×L 100 mM Tris-HCl (pH 7.9 at 25oC) 100 mM MgCl2 10 mM Dithiothreitol Medium salt buffer 10×M 100 mM Tris-HCl (pH 7.9 at 25oC) 100 mM MgCl2 500 mM NaCl 10 mM Dithiothreitol High salt buffer 10×H 500 mM Tris-HCl (pH 7.9 at 25) 100 mM MgCl2 1000 mM NaCl 10 mM Dithiothreitol Super High salt buffer 10×SH 100 mM Tris-HCl (pH 7.9 at 25oC) 100 mM MgCl2 1500 mM NaCl 10 mM Dithiothreitol Tris acetate buffer 10×A 200 mM Tris-acetate (pH 7.9 at 25oC) 100 mM Mg-acetate 500 mM K-acetate 10 mM Dithiothreitol The activity of each restriction enzyme was evaluated in each of the above five buffers containing 100µg/ml bovine serum albumin (BSA). Some SBS restriction endoncleases require Triton X-100 (TX-100). This means that 100% of the activity documented is obtained using this additive. Nineteen SBS enzymes- Asu II, BamH I, Bgl I, BseA I, BsiS I, BssA I BstE II, CspA I, EcoR I, Kpn I, Mbo I, Nco I, Not I, Nru I, Pst I, Sca I, SgrB I, SnaB I and Taq I require unique (U) buffers for optimal reaction conditions. The composition of each unique buffer is presented in specific restriction endonuclease descriptions, also in the Technical Data Sheet provided with each enzyme. Note: SBS buffers should be thawed completely and mixed thoroughly before use. Recommended Reaction Conditions for SBS Restriction Endonucleases * Requires Triton X-100 for optimal activity. Relative Activity of Restriction Enzymes with SBS Buffers This table lists relative activities of each restriction enzyme with each buffer assuming the activity of the enzyme under optimal conditions to be 100%. Reactions were carried out at 37oC except for Bcl I, BseA I, BseB I, BseC I, BsiS I, BssA I, BstE II, PspP I, Sfi I, Sma I and Taq I. The reaction temperature for these enzymes is indicated in parenthesis. All reactions were carried out in the presence of BSA, 100µg/ml. Double Digestion of DNA with SBS enzymes Simultaneous cleavage of DNA with two different restriction endonucleases is a common time-saving procedure. The digestion buffer can be chosen following the recommendations of SBS Enzyme Activity Chart which rates the activity of each restriction endonuclease in the five SBS buffers. In the table below you will find recommended buffers for double digestion using 17 of the most common restriction endonucleases. If no single SBS buffer can be found to satisfy the buffer requirements of both enzymes, the reactions can be done sequentially (seq). First, cleave with the restriction endonuclease that requies the lower salt reaction conditions, then adjust the salt concentration to complete the second reaction. When using restriction endonucleases in non-optimal SBS buffers, more enzyme or longer digestion time may be needed to compensate for the slower rate of cleavage under those conditions. Suggested SBS Buffer for Double Digestion Notes: All the reactions were carried out in the presence of BSA (100µg/ml). Our experience indicates that it is important to use BSA in reaction mixtures in order to obtain successful digestions of DNA. The presence of BSA gives complete and reproducible cleavages for a range of DNA substrates. BSA stabilizes the enzymes when digestions are perfomed for more than one hour at 37oC, since many restriction endonucleases in reaction buffers without BSA can survive at this temperature for 10-20 minutes only or even less. Also, BSA binds metal ions, and other chemicals, which might be present in buffers or DNA preparations, thereby inactivating restriction endonucleases. The following enzymes can exhibit "star" activity: BamH I, Bcl I, BseB I, BssA I, EcoR I, EcoR V, Hind III, Hpa I, Kpn I, Nco I, Nru I, Pst I, Pvu II, Sal I, Sca I, SnaB I, Ssp I, Xba I. Ligation and Recleavage with SBS Restriction Enzymes Enzyme Ligation and Recleavage Alu I >95% after 10-fold overdigestion ApaL I >98% after 100-fold overdigestion Asu II (BspT104 I) >95% after 50-fold overdigestion BamH I >95% after 10-fold overdigestion Bcl I (Fba I) >95% after 50-fold overdigestion Bgl I >95% after 50-fold overdigestion Bgl II >95% after 50-fold overdigestion BseA I (BspM II, Aor13H I) >98% after 100-fold overdigestion BseB I (BstN I) <50% after 10-fold overdigestion BseC I (Cla I) >95% after 100-fold overdigestion BshF I (Hae III) >95% after 50-fold overdigestion BsiS I (Hpa II, Msp I) >95% after 50-fold overdigestion BssA I (Cfr10 I) >95% after 30-fold overdigestion BstE II (BstP I) >95% after 100-fold overdigestion CspA I (Age I) >90% after 10-fold overdigestion EcoR I >98% after 50-fold overdigestion EcoR V >95% after 20-fold overdigestion Hind III >98% after 100-fold overdigestion Hinf I >90% after 10-fold overdigestion Hpa I >95% after 10-fold overdigestion Kpn I >95% after 10-fold overdigestion Mbo I (Sau3A I) >95% after 10-fold overdigestion MspC I (Afl II) >95% after 10-fold overdigestion Nae I >80% after 10-fold overdigestion Nco I >95% after 10-fold overdigestion Nhe I >98% after 50-fold overdigestion Not I >98% after 30-fold overdigestion Nru I <20% after 10-fold overdigestion PspP I (Sau96 I, Cfr13 I) >95% after 10-fold overdigestion Pst I >95% after 10-fold overdigestion Pvu II >95% after 10-fold overdigestion Rsa I >95% after 10-fold overdigestion Sal I >95% after 10-fold overdigestion Sau3A I (isoschizomer) >95% after 10-fold overdigestion Sca I >90% after 10-fold overdigestion Sfi I >95% after 10-fold overdigestion SgrB I (SacII) >98% after 10-fold overdigestion Sla I (Xho I) >98% after 10-fold overdigestion Sma I >95% after 10-fold overdigestion SnaB I >98% after 10-fold overdigestion Sph I >98% after 10-fold overdigestion SseB I (Stu I) >95% after 10-fold overdigestion Ssp I >95% after 10-fold overdigestion Sst I (Sac I) >95% after 10-fold overdigestion Sty I (EcoT14 I) >98% after 50-fold overdigestion Taq I >90% after 50-fold overdigestion Xba I >98% after 100-fold overdigestion Restriction Endonuclease Troubleshooting Guide Problem Probable Causes Recommended Solutions Undigested or incompletely digested DNA Nature of DNA The activity of enzyme depends on the nature of DNA substrate-linear or supercoiled, concentration of recognition sequences, origin of nucleotides flanking the recognition sequence. Verify the activity of enzyme using the substrate indicated in the product description. If the activity is indicated correctly use more enzyme in order to digest the particular substrate DNA. Impure DNA DNA preparations, especially minipreps, may contain some contaminants such as phenol, chloroform, ethanol, detergents, EDTA, salts, that might partially or completely inhibit the activity of restriction endonuclease. If the enzyme cleaves poorly particular substrate, check the activity of enzyme on unit substrate DNA (λ, Adenovirus-2, pBR322) as well as on the particular DNA mixed with unit substrate DNA. If the activity on unit substrate DNA alone corresponds to that indicated on the enzyme Technical Data Sheet whereas unit substrate DNA mixed with the DNA of interest is digested poorly, repurify the DNA. Lack of recognition sequences Check if the DNA sequences recognized by that particular endonuclease are really present in the DNA substrate tested. Nonoptimal reaction conditions Enzymatic reaction is characterized by following conditions: incubation temperature, buffer ionic strength and pH, Mg2+ concentration. Sometimes the presence of BSA in the reaction mix has the crucial influence on the activity of enzyme, because it stabilizes the enzyme, binds some impurities, prevents the enzyme sorption on the test tube surface. Reaction conditions for each endonuclease are indicated by the producing company on the Technical Data Sheet. If it is possible use the reaction buffer recommended by the producer and usually provided with the enzyme. Sometimes the low activity of enzyme might be due to the decay of reaction buffer, in such cases prepare a fresh buffer. Enzyme sensitivity to substrate methylation Each restriction endonuclease is inhibited by the methylation of one particular nucleotide in the recognition sequence. The methylation of other nucleotides in the target sequence might partially or completely inhibit the activity of the enzyme. Most laboratory strains used for plasmid preps contain two site-specific DNA methylases: Dam and Dcm. Enzyme sensitivity to dam or dcm is indicated in the catalog in the product description. Mix DNA of interest and unit substrate DNA, then digest both with the particular enzyme. When the DNA of interest only is digested poorly check if the DNA has any methylation and if the restriction endonuclease used in experiments is sensitive to that methylation. Improper dilution or addition of enzyme SBS recommends the following dilution buffer: 20mM KPO4 (pH 7.4), 200mM KCl, 1mM EDTA, 7mM β-mercaptoethanol, 10% glycerol and 0.2mg/ml BSA. The enzyme after dilution with this buffer should be used the same day. In order to store the diluted enzyme the enzyme storage buffer should be used for dilution. Not dilute the enzyme with 1 × reaction buffer. Such dilution might partially or completely inactivate the enzyme, especially if the reaction buffer has low ionic strength and no stabilizing agents. Restriction endonuclease is always added the last to the reaction mix. High glycerol concentration Some resriction endonucleases (Nco I, Hpa I) are very sensitive to glycerol concentration in the reaction mix. If the resriction enzyme is supplied in 50% glycerol it should make not more than 1/10 of the final reaction volume. Partially or completely inactivated enzyme If the reaction conditions and the substrate completely correspond to those indicated on the Technical Data Sheet and the enzyme still gives no cleavage or its activity is lower than indicated that means that due to improper handling the enzyme got inactivated. Additional bands untypical for the banding pattern of particular substrate Incomplete digestion Sometimes it is difficult to distinguish between the untypical banding pattern and incomplete digestion. In the case of partial digestion additional low intensity bands are higher than normal ones and there should be no additional bands below the smallest expected fragment. The bands disappear when the incubation time or the amount of enzymes is increased. Use 5- to 10-fold excess restriction enzyme. Enzyme "star" activity Some restriction endonucleases under particular conditions (low ionic strength, high pH of the reaction buffer, high concentration, Mn2+ instead of Mg2+, organic solvents) relax their substrate specificity-in addition to the normally recognized sequences they begin to cleave the substrate in the other sites. Additional DNA bands in such case should be lower than the expected bands and there should be no additional bands higher than the biggest expected fragment. In the case of star activity, additional bands increase and the typical banding pattern decreases in intensity when more enzyme is added, or the incubation time is prolonged. In order to avoid the star activity lower excess amount of enzyme should be added (no more than 10-fold), reaction should be performed in the buffer (sometimes designed specially to suppress the star activity) recommended by the producer. The enzyme is contaminated with other restriction endonuclease If following all the above recommendations the incorrect banding pattern still persists, especially if smaller than expected additional bands are obtained, the enzyme preparation due to improper handling might got contaminated with another restriction endonuclease. Sometimes not the enzyme preparation might contain contaminating endonuclease but the reaction buffer, DNA preparation or the stop solution. Detect second activity by comparing restriction digest pattern to that expected for the DNA used for unit determination. DNA preparation tested contaminated with another DNA Detect by DNA minus enzyme on gel; digest the DNA with other restriction endonucleases. Diffuse DNA bands after gel electrophoresis band shift Impure substrate Incubate the substrate DNA without the enzyme and with the other restriction endonuclease for control. Impure enzyme Verify the activity of enzyme using the substrate indicated in the product description. Diffusion bands in such case indicate that the enzyme got contaminated due to improper handling. Protein binding to DNA results in the gel shift in agarose gels Incubate the samples before the electrophoresis with 0.1% SDS (final concentration) at 65oC for 10 minutes. Poor ligation efficiency Decay of the buffer components ATP and DTT included in the ligation buffer are easily degraded substances. Repeat the ligation with fresh ligation buffer. Restriction enzyme still active in the ligation mix If the restriction endonuclease is not heat inactivated at 65oC (thermostable), phenol deproteinization and/or ethanol precipitation of the fragments after the restriction reaction should be performed. Nonspecific nuclease contamination Restriction endonuclease, Ligase, or DNA preparation might contain nonspecific nucleases due to improper handling in the lab. Figure out which component of the ligation mix is contaminated by substituting them one by one. Ligase concentration is too low In the blunt end DNA ligation reactions the concentration of ligase in the reaction mix should be 1.5-7.5 Wu/ml. DNA fragments obtained after cleavage with following restriction endonucleases: BseB I, Nru I are ligated very poorly. In such cases high amounts of Ligase (0.3-0.6 Wu/µg of fragments) and 10% PEG in the ligation reaction should be used. DNA phenol deproternization after the restriction reaction should be performed. Viswagen Biotech Pvt. Ltd., 23/863G, Thazhathuveettil Buildings, Market Road, Pala, Kerala 686 575; India Web: www.viswagen.com, Phone: +91-98-46804374; Fax: +91-4822-215075 Contacts: customer service: customer.service@viswagen.com Copyright © 2007 Viswagen Biotech Pvt. Ltd.