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