In extreme cases the dna can be precipitated after one digest and dissolved in the second digest buffer. It is also used to quickly check the identity of a plasmid by diagnostic digest. Dna digestion and electrophoresis in this experiment we will be doing a process called as dna digestion or also known as restriction digest. A basic protocol for use of promega restriction enzymes. 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. We compared restriction enzyme analysis of plasmid reap dna profiling with bacteriophage typing for determination of similarities and differences among 50 pairs of staphylococcus aureus blood isolates from patients with multiple positive blood cultures. This is important so that when bacteria replicate, the plasmid is. However,the majority of enzymes make cuts staggered on each strand, resulting in a few base pairs of single stranded dna at each end of the fragment, known as sticky ends. Some enzymes create 5 overhangs and others create 3 overhangs. Experiment 2 plasmid dna isolation, restriction digestion.
Dna restriction digestion analysis teacher s guide book cat. This obviously doubles the time required for digestion. A rapid and simple plasmid isolation procedure was developed for the epidemiological analysis of plasmidmediated antimicrobial resistance. A restriction digest is a procedure used in molecular biology to prepare dna for analysis or other processing. Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave dna at specific sequences. Restriction enzyme digestion is commonly used in molecular cloning techniques, such as pcr or restriction cloning. Isolates from 17 pairs did not have detectable plasmids. Restriction enzyme protocol pdf promega corporation. Dna is eluted during incubation at 65oc and then removed from the particles. Restriction endonuclease digestion of plasmid dna essay. The mcs is the site on a plasmid where new dna fragments are inserted. Restriction enzyme lab report essay example graduateway. For a list of many commonly used restriction enzymes, visit neb. If a hybrid recombinant plasmid was constructed from pbr325 by the insertion of a fragment of dna at the bamhi restriction site, firstly the total size of the plasmid got bigger.
We will also perform analysis of the purified plasmid. If two pieces of dna have complementary sticky ends, they. Measure the volume of the aqueous dna solution and mix gently with 10% vv 3 m naacetate, ph 5. Plasmid dna isolation, restriction digestion and gel electrophoresis plasmid dna isolation introduction. In this exercise, you will digest the plasmid pbr322 with 5 different restriction enzymes and resolve the fragments by agarose gel electrophoresis.
Most restriction enzymes res will not cut dna that is methylated on one or both strands of their recognition. A recombinant plasmid pww1 with complicated structure was shown by endonucleases digestion analysis, indicated that a possible rearrangement of pseudomonas aeruginosa dna. In extreme cases the dna can be precipitated after one digest and. Restriction digests are mixtures of dna fragments produced by the reaction of dna. It is an alteration of the specificity of restriction enzyme mediated cleavage of dna that can occur under some non standard conditions that differ from the optimum for. Protocol for dna digestion with a single restriction enzyme.
Experiment 2 plasmid dna isolation, restriction digestion and gel. A specific protocol for single digestion using this restriction enzyme can be accessed using our free online tool. Contaminating nucleases are usually activated only after the addition of salts e. Figure 1 restriction map of y1p5, a 5,541 base pair plasmid.
Restriction digestion the idea a restriction digest is used to cut dna at specific sequences to leave sticky ends. Protocol pbr322 restriction analysis of plasmid dna. Digest dna plasmids with unique restriction enzymes. Anza restriction enzymes show complete digestion in 15 minutes with no star activity after overnight digestion.
Diagnostic digestion of plasmid dna restriction enzyme digestions and mapping of dna fragments background. There are hundreds of different restriction enzymes, allowing scientists to target a wide variety of recognition sequences. Plasmid dna isolation and restriction enzyme digests. Restriction analysis of plasmid dna in this exercise, you will digest the plasmid pbr322 with 5 different restriction enzymes and resolve the fragments by agarose gel electrophoresis. Plasmid dna is used for a number of downstream applications such as transfection, sequencing, screening clones, restriction digestion, cloning, and pcr. Prepare negative control reaction without template dna.
Dna restriction digests and agarose gel electrophoresis. Restriction digestion involves fragmenting dna molecules. Purified plasmid dna is digested with 1 or more restriction enzymes res selected to give a distinct dna band pattern that is easily resolved by electrophoresis. We compared restriction enzyme analysis of plasmid reap dna profiling with bacteriophage typing for determination of similarities and differences among 50 pairs of. 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. Methylation of restriction endonuclease sites would have made plasmid molecules resistant to the specific endonuclease mcq. Restriction digestion involves fragmenting dna molecules into smaller pieces with special enzymes called restriction endonucleases commonly known as restriction enzymes re. Unless directed otherwise, keep all tubes on ice at all times. Molecular biology protocol restriction digest of plasmid dna. In all cases, one or more restriction enzymes are used to digest the dna resulting in either nondirectional or directional insertion into the compatible plasmid. Restriction endonuclease digestion of plasmid dna 1. Restriction enzyme digestion principle shomus biology.
To perform a rapid digestion, assemble the following components on ice in 0. 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. All restriction enzymes cut dna between the carbon and the phosphate moiety of the phosphodiester bond so that fragments produced by restriction enzyme digestion have. Prepare positive control reaction with template of known cutting site corresponding to the restriction enzyme of choice. This dna will run faster on a gel and is resistant to restriction enzyme digestion. This lab will introduce you to dna modification by restriction enzymes using the purified plasmids you prepared from your transformation. Digestion with restriction enzyme addition of foreign dna ligase plasmid without insert plasmid with insert cloned dna fragment open plasmid isolation of plasmid dna to.
Methylation of specific adenine or cytidine residues within the recognition sequence of the restriction enzyme affects the digestion of dna. It is sometimes termed dna fragmentation this term is used for other procedures. Restriction enzymes digestionrestriction endonuclease. Restriction digest of plasmid dna flashcards quizlet. Restriction digestion principle pdf to perform restriction digestion of lambda. Long exposure to alkaline conditions may cause the plasmid dna to become irreversibly denatured. The dna can be run on an agarose gel to visualize the dna or can be subjected to restriction digestion analysis and. The procedure for restriction cloning is quite simple. Learn to perform digestions with restriction enzymes. Learn vocabulary, terms, and more with flashcards, games, and other study tools. The application of molecular biology techniques to the analysis of complex. Cloning by restriction enzyme digestion and ligation is a simple and easy way of moving a fragment of doublestranded dna from one plasmid to another. Restriction digest protocol a specific protocol for single digestion using this restriction enzyme can be accessed using our free online tool, nebcloner.
Digests are carried out at 37 degrees c unless otherwise noted for the enzyme. First quantify the plasmid by gel comparison, not nanodrop. Robert weinberger, in practical capillary electrophoresis second edition, 2000. Restriction digestion also called restriction endonuclease is a process in which dna is cut at specific sites, dictated by the surrounding dna sequence. Extracting the backbone plasmid backbones are separated and extracted by dna gel agarose electrophoresis the gel separates the dna based on the size number of. In this experiment, using agarose gel electrophoresis, the number and relative positions of restriction sites for three restriction enzymes, ecor1, hincii and pvuii, on the circular plasmid. Restriction enzymes digest the plasmid, you prepare an insert either from another plasmid or one you synthesized, and. Table 1 protocol for the cutting of plasmid dna with. To purify plasmid dna from a bacterial culture to learn to measure small volumes of liquids using micropipettes to prepare competent cells and conduct transformation with plasmid dna to. Restriction digest an overview sciencedirect topics.
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