There are hundreds of different restriction enzymes, allowing scientists to target a wide variety of recognition sequences. Robert weinberger, in practical capillary electrophoresis second edition, 2000. In extreme cases the dna can be precipitated after one digest and dissolved in the second digest buffer. Molecular biology protocol restriction digest of plasmid dna. Prepare negative control reaction without template dna. Prepare positive control reaction with template of known cutting site corresponding to the restriction enzyme of choice.
Restriction digestion involves fragmenting dna molecules. Restriction enzymes digestionrestriction endonuclease. Dna restriction digestion analysis teacher s guide book cat. Dna restriction digests and agarose gel electrophoresis. Protocol for dna digestion with a single restriction enzyme. Restriction digest of plasmid dna flashcards quizlet. Experiment 2 plasmid dna isolation, restriction digestion. It is sometimes termed dna fragmentation this term is used for other procedures. Contaminating nucleases are usually activated only after the addition of salts e. Protocol pbr322 restriction analysis of plasmid dna. Diagnostic digestion of plasmid dna restriction enzyme digestions and mapping of dna fragments background.
A specific protocol for single digestion using this restriction enzyme can be accessed using our free online tool. 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. Dna is eluted during incubation at 65oc and then removed from the particles. 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. Long exposure to alkaline conditions may cause the plasmid dna to become irreversibly denatured. 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. Learn to perform digestions with restriction enzymes. First quantify the plasmid by gel comparison, not nanodrop. Digest dna plasmids with unique restriction enzymes. 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.
This obviously doubles the time required for digestion. Isolates from 17 pairs did not have detectable plasmids. The mcs is the site on a plasmid where new dna fragments are inserted. Experiment 2 plasmid dna isolation, restriction digestion and gel. We compared restriction enzyme analysis of plasmid reap dna profiling with bacteriophage typing for determination of similarities and differences among 50 pairs of. Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave dna at specific sequences. Methylation of specific adenine or cytidine residues within the recognition sequence of the restriction enzyme affects the digestion of dna. For a list of many commonly used restriction enzymes, visit neb. Plasmid dna isolation and restriction enzyme digests. If two pieces of dna have complementary sticky ends, they. Restriction endonuclease digestion of plasmid dna 1. In extreme cases the dna can be precipitated after one digest and. Restriction enzyme digestion principle shomus biology.
It is also used to quickly check the identity of a plasmid by diagnostic digest. Restriction enzyme protocol pdf promega corporation. 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. We will also perform analysis of the purified plasmid. A restriction digest is a procedure used in molecular biology to prepare dna for analysis or other processing. In this exercise, you will digest the plasmid pbr322 with 5 different restriction enzymes and resolve the fragments by agarose gel electrophoresis. To perform a rapid digestion, assemble the following components on ice in 0.
Table 1 protocol for the cutting of plasmid dna with. This is important so that when bacteria replicate, the plasmid is. Restriction enzyme digestion is commonly used in molecular cloning techniques, such as pcr or restriction cloning. Most restriction enzymes res will not cut dna that is methylated on one or both strands of their recognition. Restriction digests are mixtures of dna fragments produced by the reaction of dna. 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. Restriction endonuclease digestion of plasmid dna essay. Protocols for rapid digestion of plasmid dna in 515 minutes and for direct digestion of pcr or. The dna can be run on an agarose gel to visualize the dna or can be subjected to restriction digestion analysis and. A recombinant plasmid pww1 with complicated structure was shown by endonucleases digestion analysis, indicated that a possible rearrangement of pseudomonas aeruginosa dna. This lab will introduce you to dna modification by restriction enzymes using the purified plasmids you prepared from your transformation.
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. Unless directed otherwise, keep all tubes on ice at all times. Digests are carried out at 37 degrees c unless otherwise noted for the enzyme. Some enzymes create 5 overhangs and others create 3 overhangs. Anza restriction enzymes show complete digestion in 15 minutes with no star activity after overnight digestion. Plasmid dna is used for a number of downstream applications such as transfection, sequencing, screening clones, restriction digestion, cloning, and pcr. 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 digest protocol a specific protocol for single digestion using this restriction enzyme can be accessed using our free online tool, nebcloner.
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. 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. Figure 1 restriction map of y1p5, a 5,541 base pair plasmid. The application of molecular biology techniques to the analysis of complex. 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 digestion principle pdf to perform restriction digestion of lambda. Restriction digest an overview sciencedirect topics. 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.
Measure the volume of the aqueous dna solution and mix gently with 10% vv 3 m naacetate, ph 5. Restriction enzymes digest the plasmid, you prepare an insert either from another plasmid or one you synthesized, and. Plasmid dna isolation, restriction digestion and gel electrophoresis plasmid dna isolation introduction. Methylation of restriction endonuclease sites would have made plasmid molecules resistant to the specific endonuclease mcq. The number after each restriction enzyme name indicates at which base pair the dna is cut by that enzyme. Restriction digestion also called restriction endonuclease is a process in which dna is cut at specific sites, dictated by the surrounding dna sequence. 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.
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