We will use the genomic dna and the plasmid dna that you. The dna restriction digestion analysis kit demonstrates the specificity of restriction enzymes and their need for specific buffers. Restriction digestion of dna practical kit guide pdf. Dna restriction digests and agarose gel electrophoresis. Hydrolysis of nucleic acids by cathepsin d or trypsin. Prepare positive control reaction with template of known cutting site corresponding to the restriction enzyme of choice.
Digestion of dna with restriction endonucleases bloch 1995. For many dna manipulations such as restriction enzyme analysis, subcloning and agarose gel electrophoresis, the simple methods are sufficient. The gel is then subjected to an electric field, which draws the negatively charged dna across it. You will be using restriction endonuclease enzymes that recognize and. Typical incubation time and temperature is 37c for 1 hour, though time and temperature will vary depending on restriction enzyme used. Jul 14, 2015 hydrolysis of nucleic acids by cathepsin d or trypsin. A restriction digest is a procedure used in molecular biology to prepare dna for analysis or other processing. This paper is concerned with only classit restriction enzymes. Restriction analysis links to biotechnology the techniques introduced in this exercise form the basis of recombinant dna technology techniques, eukaryotic and prokaryotic gene structure dna fingerprinting, and forensic dna analysis. In this study, the effects of dna sequence specificity, purine content ag content. Restriction enzyme troubleshooting guide thermo fisher.
Watch the video below to learn how to analyze your restriction digest results. If you want to digest 1 ul of dna with a concentration of 5ugul, you should make a 1. To digest dna you combine dna, enzyme, and buffer in a tube, and incubate for a period of time at a specific temperature. Dna fingerprinting by restriction enzyme digestion of dna introduction in this exercise you will be learning how variation in dna sequence can be used to distinguish between individual dna molecules.
Dna by cathepsin d or trypsin was similar to the protocol used for pepsin. The first part of this practical covered restriction digestion of a plasmid dna with various enzymes and separation of the resulting dna fragments by electrophoresis on an agarose gel. Prepare negative control reaction without template dna. This lab introduces the analysis of dna by restriction digest and gel electrophoresis using plasmid dna info on dna provided by instructor at time of laboratory. Dna is digested in separate reactions with one of up to three. Restriction enzymes, also referred to as restriction endonucleases, are enzymes that recognize short, specific often palindromic dna sequences. Add the following reaction components in the order indicated. Restriction digestion and analysis of lambda dna kit.
Before starting the experiment, crush ice and place the vials containing lambda dna, restriction enzymes and assay buffers onto it. How do i calculate how much to use in a digestion of 1ug of dna. Restriction endonuclease cleavage of dna into discrete fragments is one of the most basic procedures in molecular biology. Some restriction enzymes may display star activity, a less stringent sequence dependence that results in altered specificity and more randomly digested dna. Restriction endonuclease bsti cleaves dna in a position identical with that. Comparison of analytical and predicted dna digestion patterns indicates presenceabsence of restriction sites at the expected position within the plasmid construct providing some information about the insert sequence. Therefore, appropriate control reactions should always be run in parallel with the restriction enzyme digest see section 5. An extension of this allows students to understand the crucial role restriction. Oct 16, 2012 this video demonstrates how to set up a restriction digest using the biorad restriction digestion and analysis of lambda dna kit.
Ct sites in your sequence and record the number of times alui will cut your dna. Dna fingerprinting by restriction endonuclease digestion. Preparation of dna for traditional cloning methods is dependent upon restriction enzyme digestion to generate compatible ends capable of being ligated together. Dna restriction and gel electrophoresis this week, we will learn how restriction enzymes can be used to evaluate genomic and plasmid dna. Restriction enzymes are nucleases which can cleave the sugarphosphate backbone of dna, found in bacteria. First quantify the plasmid by gel comparison, not nanodrop. There is no universal digestion buffer for restriction enzymes as different enzymes have different preferences for. Dna molecule has a double helix structure with two strands of nucleotides coiled together and held in place by a 2deoxyribose sugarphosphate backbone. Restriction digestion of plasmid dna free download as powerpoint presentation. This video demonstrates how to set up a restriction digest using the biorad restriction digestion and analysis of lambda dna kit. Using this ratio, you can calculate the minimal amount of enzyme for your reaction.
Open the modules immediately upon receipt and store components at 20c, 4c, or room temperature as indicated. Recently, the proteinspecific enzyme pepsin was found be capable of digesting nucleic acids unexpectedly. As they cut within the molecule, they are commonly called restriction endonucleases. Therefore, pcr reaction mixture should not make more than volume of digestion reaction mixture to avoid inhibitory effects. Table 1 protocol for the cutting of plasmid dna with. A restriction enzyme digest should be carefully planned. Scientists discovered restriction enzymes in 1962 while. The dna to be cloned can vary widely, from genomic dna extracted from a pure bacterial culture or a mixed population, to a previously cloned gene that needs to be moved from one vector. A control uncut sample is incubated without adding enzyme. There is no universal digestion buffer for restriction enzymes as different enzymes have different preferences for ionic strength salt concentration and major cations sodium or potassium.
Digested dna samples are loaded into wells of an 0. To understand the method of digesting dna with different restriction enzymes. In gel electrophoresis, a sample of dna is first loaded onto a slab of agarose gel literally pipetted into small wells at one end of the slab. Techniques for setting up a restriction digest using a. Comparison of analytical and predicted dna digestion patterns indicates presenceabsence of restriction sites at the expected position within the plasmid construct providing some information about the. Dna digestion by restriction enzymes can be a sensitive process dependent on the concentrations of the reactions components and reaction time. Our restriction enzyme collection has been optimized for digestion using five unique buffers. There are 3 4 commonly used buffers that are generally suitable for most enzyme conditions. In this experiment, lambda dna is digested with two restriction. A digestion reaction typically consists of the following. Techniques for setting up a restriction digest using a micropipet. The ability to cleave dna at specific sites is one of the cornerstones of todays methods of dna manipulation. Today dna research has revealed intricate details of how the genetic code is copied or duplicated during cell division.
Ecori is a type ii restriction enzyme, isolated from e coli, that recognises and cuts the sequence. Find more protocols and selection guides in the molecular biology guide. In this experiment, lambda dna is digested with two restriction enzymes. Dna methylation has an inhibitory effect on some restriction enzymes. Restriction enzyme digestion is commonly used in molecular cloning techniques, such as pcr or restriction cloning. Restriction enzymes play a very important role in the construction of recombinant. Students calculate the size of expected fragments from a linear map of pbr322 figure 2 and compare these with the experi. There is no universal digestion buffer for restriction enzymes as different enzymes have different preferences for i onic strength salt concentration and major cations sodium or potassium. When digesting dna using a single enzyme, use the buffer supplied with the enzyme also identified on table 1 of the restriction enzyme buffer reference. Dna, restriction enzymes, and gel electrophoresis introduction in this twoday lab you will explore the many properties of dna. Restriction endonucleases, or restriction enzymes, are important classes of enzymes used by biotechnologists.
Experiment 2 plasmid dna isolation, restriction digestion and gel. The enzyme will cleave cut the dna fragment every time gaattc is found in the sequence. Restriction enzyme protocol pdf promega corporation. For digestion of unpurified pcr products, set up the digestion reaction with the pcr mixture being no more than of the final reaction volume e.
Dna fingerprinting by restriction endonuclease digestion an assault in east lansing you are currently employed by the michigan state police as a forensic dna analyst at their lansing laboratory. These special enzymes recognize specific sequences in the dna molecule for example gatatc wherever that sequence occurs in the dna. Restriction digestion is the process of cutting dna molecules into smaller pieces with special enzymes called restriction endonucleases sometimes just called restriction enzymes or res. This process is monitored and controlled by the dna replication enzymes. A protocol for methylating specific dna sequences and protecting them from restriction endonuclease cleavage is also presented. Contaminating nucleases are usually activated only after the addition of salts e.
Digestion of pcr products thermo fisher scientific. Recombinant dna refers to the creation of new combinations of dna segments that. Application note l01 restriction digestion of lambda dna. When dna is cut, the resulting fragments are called restriction fragments or rflps. An extension of this allows students to understand the crucial role restriction enzymes play in molecular cloning and the analysis of genomic and recombinant dna. The amounts of dna, enzyme, buffer and water must be determined before you begin pipetting. The enzyme binds and cuts dna between the guanine and cytosine bases. However, digestion of pcr products in the amplification mixture is often inefficient. Dna fingerprinting by restriction enzyme digestion of dna introduction in this exercise you will be learning how variation in dna sequence can be used to distinguish between individual dna molecules and therefore between individual organisms. A fragment of dna in solution is treated with a specified restriction endonuclease in a process called restriction digest. We have examined the reaction of the ecori restriction endonuclease on dna. Dna fragments r to construct a restric tioh map with data obtained from gel electrophoresis. In gel electrophoresis, a sample of dna is first loaded onto a slab of agarose gel literally pipetted into.
Mechanism of extraordinary dna digestion by pepsin. The fluorescent bands of the dna restriction fragments in lane 2, 3, 4, and 5 were several fold brighter than the uncut plasmid band in lane 7. To investigate the efficiency and outcome of cutting singledigested lambda dna with the restriction enzyme ecori, using wealtecs cb1 block cooler as incubation system. Two important applications are dna fingerprinting and methylation analysis, which are methods to map sequences and analyze epigenetic patterns in the genome. Restriction digestion is a necessary process to be able to perform analysis of the dna by rflp, aflp or strp techniques. Almost all strains of li contain two site specific dna methylases that methylate specific sequence s. It is also used to quickly check the identity of a plasmid by diagnostic digest.
Incomplete digestion or no digestion possible cause recommended solution if the enzyme doesnt cut the control dna. Purification, properties and specificity of the restriction. Check the expiration date verify that the enzyme has been stored at 20c check the temperature of your freezer. Protip the amount of restriction enzyme you use for a given digestion will depend on the amount of dna you want to cut. Restriction enzymes are typically inactivated by incubation at high temperature. One example is treatment of a 5,000 base pair 5kb fragment with ecor1. Publisher summary this chapter discusses the interaction of restriction endonucleases with doublestranded dna molecules at specific sites leading to cleavage of the dna into a number of fragments. Duplication of any part of this document is permitted for classroom. Restriction analysis of lambda dna terrific science. Recombinant dna technology development and applications b. To investigate the efficiency and outcome of cutting singledigested lambdadna with the restriction enzyme ecori, using wealtecs cb1 block cooler as incubation system.
Setting up restriction enzyme digests setting up enzyme digests is simple if you follow a few rules and guidelines. Although restriction enzymes are widely used in molecular cloning, their use as molecular tools extends to other common applications in molecular biology. To digest dna you combine dna, enzyme, and buffer in a tube, and incubate for a. Learn to perform digestions with restriction enzymes. Restriction enzymes in genome mapping and analysis thermo. One example is treatment of a 5,000 base pair 5kb fragment with. Incubation time and temperature is 65c for 20 min, though time and temperature will vary depending on restriction enzyme used. After restriction digest, dna can then be analysed using agarose gel electrophoresis. A new case has just come in this morning requiring your considerable expertise.
On the first day you will use salmon sperm dna to demonstrate. On the first day you will use salmon sperm dna to demonstrate the properties of denaturation and renaturation. Next you will perform extractions of dna from both salmon sperm and e. If there ware, they must be the chromosomal dna bands, the absent of these in lane 2, 3, 4 and 5 was because they were too small, might not be seen, or ran off the gel. To perform restriction digestion of dna with ecor i and bamhi enzymes. To investigate the efficiency and outcome of cutting singledigested lambdadna with the restriction enzyme ecori, using wealtecs cb. In addition, plasmid size can be confirmed by simply adding the size of each dna band in the digestion. Dna loading buffer ifelectrophoresis issubsequent agarose gel 0. Restriction digestion of plasmid dna plasmid restriction. Restriction enzymes play a very important role in the construction of recombinant dna molecules, as is done in gene cloning experiments.
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