How much restriction enzyme buffer to use

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Explore what makes good science possible. PCR products in which the first base pair of the restriction site was flush with 0 , or 1, 2, or 3 base pairs away from the end of the fragment were tested with a variety of enzymes. Purified PCR fragments ng were digested at least twice with 0. Table reproduced by permission of Eaton Publishing. The addition of upstream bases to PCR primers is not the only method used to improve digestion efficiency.

A number of protocols have been proposed to improve digestion including proteinase K treatment to remove any thermostable polymerase that may be blocking the DNA, end-polishing with Klenow or T4 DNA Polymerase and the addition of spermidine. However, none of these methods have been shown to improve cloning efficiency significantly 4 5.

An additional drawback to the incorporation of restriction enzyme sites in PCR primers is that it can be quite difficult to resolve digested PCR products from those that remain uncut. This allows identification of products that have been cut successfully because the label is lost upon digestion 6. An alternative method that has been used successfully to improve digestion of PCR products is to concatemerize the fragments after amplification 1 5.

This is achieved by first treating the cleaned up PCR products with T4 Polynucleotide Kinase if the primers have not already been phosphorylated. The ends will already be blunt if a proofreading thermostable polymerase such as Pfu was used or may be treated with T4 DNA Polymerase to polish the ends if a non-proofreading polymerase such as Taq was used.

This effectively moves the restriction enzyme sites away from the ends of the fragments and allows efficient digestion. This troubleshooting guide addresses common problems that may be encountered while using restriction enzymes.

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Explore Support Center Medical Affairs We provide medical information and facilitate research collaborations. Connect With Us. Local Sales Support Get in touch with a nearby distributor or sales representative. Find Sales Contact. Contact Us Customer Support. Home Resources Product Guides and Selectors Restriction Enzyme Resource This guide introduces restriction enzymes, providing i n-depth reference information and tools to help you find buffers for double digests, or find enzymes by name or recognition sequence.

Go to Products. History Restriction enzymes recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to these sequences.

References Roberts, R. Gene , 19— Luria, S. Bertani, G. Arber, W. Host controlled modification of bacteriophage lambda. Dussoix, D. Control over acceptance of DNA from infecting phage lambda.

Linn, S. And Arber, S. Purification and general properties. USA 59 , Meselson, M. And Yuan, R. Nature , —4. Smith, H. Kelly, T. Back to top. Restriction Enzyme Classification Restriction endonucleases are categorized into one of four general groups Types I , II , III , and homing endonucleases based on their subunit structure, cofactor requirements, specificity of cleavage, and associated methylase activity Table 1.

Recognition Sequences Most restriction endonucleases recognize palindromic or partially palindromic sites. Types and General Properties of Restriction Endonucleases The table below gives the types and general properties restriction endonucleases.

Type I EC 3. References Williams, R. Roberts, R. Kong, H. Sears, L. Nucleic Acids Res. Reuter, M. Oller, A.

Szybalski, W. Gene , 13— Belfort, M. Bickle, T. Wilson, G. Annu Rev Genet. Meisel, A. EMBO J. Kruger, D. FEMS Microbiol. References Wilson, G. Stahl, F. Bitinaite, J. Ehbrecht, H. Jeltsch, A. Robinson, C.

USA 95 , — Pingoud, A. Sam, M. Biochemistry 38 , — References Lesser, D. Science , — Vermote, C. Biochemistry 31 , —9. Type IIe Restriction Enzymes A few restriction enzymes have considerably greater difficulty in cleaving some of their recognition sites. Effector Sequences Investigation revealed that binding of a second recognition sequence, in cis or trans , to a distal, non-catalytic site on the enzyme allows slow and resistant sites to become cleavable.

References Oller, A. Reaction Conditions pH: Most restriction enzymes are used between pH 7. The following is an example of a typical analytical single restriction enzyme digestion: Under sterile conditions add the following components, in the order stated, to a sterile microcentrifuge tube.

Centrifuge briefly at 12, x g in a micro centrifuge to collect the contents at the bottom of the tube. Incubate at the optimum temperature for hours. 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. Use the optimal buffer supplied with one enzyme if the activity of the second enzyme is acceptable in that same buffer.

Be aware of possible star activity under non-optimal reaction conditions See the Reference Table Relative Activity of Restriction Enzymes in Promega 10X Buffers or use the Restriction Enzymes interactive search tool to identify compatible buffers. Choose an isoschizomer or neoschizomer with more compatible buffer requirements.

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.

Experimental Controls Some common controls used for restriction enzyme digestion and gel analysis are given in the Table below. Shows the integrity of the DNA starting material. Nicked, linear and supercoiled forms of plasmid DNA are normally seen in untreated samples. Control: No enzyme Control Strategy Purpose A mock digest is run parallel with the experimental digest, except that no enzyme is added.

The missing volume is made up with water. Compares DNA digests with and without enzyme. Detects changes that may occur independent of enzyme such as exonuclease contamination in the DNA or in one of the reaction components. Control: Enzyme activity check Strategy Purpose Perform a control digest using the unit definition DNA usually lambda and conditions as described in the Promega Product Information sheet. Confirms enzyme activity.

Control: DNA substrate control and general enzyme digest control Strategy Purpose Set up the following parallel digests: 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.

Compares activity of the enzyme under experimental conditions using standard DNA and experimental DNA under standard conditions. Tests for possible problems with substrate DNA such as impurity, missing recognition sites, methylation, etc. Can be used to assay for the function of other reagents used in the enzyme digest. The size of these fragments is measured in base pairs or kilobase bases pairs. Since the recognition site or sequence of base pairs is known for each restriction enzyme, we can use this to form a detailed analysis of the sequence of bases in specific regions of the DNA in which we are interested.

In the presence of specific DNA repair enzymes , DNA fragments will reanneal or stick themselves to other fragments with cut ends that are complimentary to their own end sequence. This DNA may contain genes that allow the organism to exhibit a new function or process.

This would include transferring genes that will result in a change in the nutritional quality of a crop or perhaps allow a plant to grow in a region that is colder than its usual preferred area. This virus is 48, base pairs in length which is very small compared with the human genome of approximately 3 billion base pairs.

If the virus DNA is exposed to the restriction enzyme for only a short time, then not every restriction site will be cut by the enzyme. This will result in fragments ranging in size from the smallest possible all sites are cut to in-between lengths some of the sites are cut to the longest no sites are cut. This is termed a partial restriction digestion. In this experiment, we will perform a full restriction digestion. After overnight digestion, the reaction is stopped by addition of a loading buffer.

The DNA fragments are separated by electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate into an agarose gel. The gel is then stained with a methylene blue stain to visualize the DNA bands and may be photographed. This laboratory will take approximately 3 days. The restriction digestion takes place overnight and can be kept in the freezer until the next class period when it will be be used for gel electrophoresis.

The gels may be stained overnight prior to photographing or recording results. Gels may be discarded in regular trash receptacle. A description of how to use a micropipet can be found in Activity 2 - Gel Electrophoresis of Dyes. Although methylene blue dye is not as sensitive as ethidium bromide it may be used to stain the higher quantities of DNA that are used in this experiment. Methylene blue is non-toxic but will stain clothes, hands, and equipment, so always wear gloves. Use the stain close to a sink and clean up spills immediately.

Use distilled or deionized water to de-stain gels. Only use deionized water for making the 0. A single container of methylene blue dye should be all that is needed since it may be reused several times and disposed of down the sink. Restriction enzymes require special care for handling and use. They lose activity unless kept frozen; exposure to warm temperatures for even a short time will result in loss of activity. Place the comb into the right position and allow it to set for approximately one hour this can be done faster by placing the gel tray in the refrigerator.

Carefully remove the comb from the gel. Rotate the gel tray so that the wells are toward the negative black terminals the top of the tank, assuming that the electrodes are on the right hand side. Cover the gel with 1X TAE running buffer. Loading the gel The samples must now be loaded into the wells in the gel left by the comb.

Load all of the loading solutions into separate wells in the gel loading the DNA ladder last into a separate well on the left or right hand side of your gel. There must be small bubbles rising from both ends of the electrophoresis chamber. Check after 5 minutes to make sure the gel is running i. Then allow the gel to run for the necessary amount of time about 1 hour however, check that the dye front has almost run through the gel. Switch off the power pack and take the gel to the transilluminator.

Take a photograph, print off and glue into your workbook. Annotate the photograph, indicating bands of interest. Pour away the buffer from the electrophoresis tank and rinse well with water.