You digest all three plasmids puc, plasmid x, plasmid y with ecori, bamhi, or both restriction enzymes together. The restriction mapping method is used for the mapping of genes on a plasmid or shorter piece of dna although mapping the entire genome using the restriction digestion or restriction method is a tedious, inaccurate and timeconsuming method. This week you will use restriction mapping to characterize the plasmid vectors containing r. Correlation of antibiotic resistance and restriction mapping. Medcram medical lectures explained clearly recommended for you. Restriction mapping the mean size is close to 4000 bp. Dna mapping lab background restriction endonucleases restriction endonucleases are enzymes that cut dna at specific sequences. Snapgene viewer includes the same rich visualization, annotation, and sharing capabilities as the fully enabled snapgene software. A restriction map is a map of known restriction sites within a sequence of dna. Using restriction mapping to teach basic skills in the.
Shmuel razin and david yogev, in molecular and diagnostic procedures in mycoplasmology, 1995. Dna fragments of this length are useful in the lab, since they long enough to contain the coding sequence for proteins and are well. A plasmid map is a graphical representation of a plasmid, which shows the locations of major landmarks or elements of the plasmid. Restriction mapping pdf at the end of this lab, students will be able to. When the plasmid dna digested with combinations of restriction enzymes and the resulting fragments are. Dna cloning with plasmid vectors molecular cell biology.
Protocol pbr322 restriction analysis of plasmid dna. Restriction enzyme digestion is commonly used in molecular cloning techniques, such as pcr or restriction cloning. Elements, name and size of the plasmid, orientation of the promoter, plasmid map, restriction enzymes, restriction mapping. Snapgene viewer is revolutionary software that allows molecular biologists to create, browse, and share richly annotated dna sequence files up to 1 gbp in length. Construction and mapping of recombinant plasmids used for the. Diagnostic digestion of plasmid dna restriction enzyme digestions and mapping of dna fragments background. Restriction mapping of plasmid a lab 1 restriction. Restriction mapping gives information about the relative position of dna sequences on a small scale 0. To insert a foreign piece of dna, the plasmid is digested with a restriction enzyme that cuts the double stranded dna at the cloning site. The restriction enzyme cuts dna at its specific location of short sequence called the restriction site.
The objective of this experiment module is to develop an understanding of. Assembly of restriction enzyme digestions promega corpora on 2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526 6082744330 fax 6082772516 1. Dec, 2014 the small size of plasmid is necessary to transfer larger sized exogenous dna. The small size of plasmid is necessary to transfer larger sized exogenous dna. In the first part, each student pair cuts a plasmid dna with one or more restriction endo nucleases and loads their sample onto one of the lanes of an. Draw a plasmid map from gel electrophoresis data of the digested recombinant plasmid. In this experiment, students will develop an understanding of. Nov 30, 2011 how i tricked my brain to like doing hard things dopamine detox duration. Restriction maps a map showing positions of restriction sites in a dna sequence if dna sequence is known then construction of restriction map is a trivial exercise in early days of molecular biology dna sequences were often unknown biologists had to solve the problem of constructing restriction maps without knowing dna sequences 6. One way to do this is to use restriction enzymes restriction endonucleases that are naturally found in bacteria to cut the dna molecule into fragments, and then perform a gel electrophoresis on the treated dna. A physical map consists of a set mof markers and a function p. The objective of this experiment module is to develop an understanding of the principles of dna mapping using. Table 1 protocol for the cutting of plasmid dna with.
Be sure to run appropriate controls on each gel, including single digests to compare with double digests or doubles to be compared with each other. Using these data, construct a restriction map of plasmid pda102 for the restriction enzymes sali and hhaiii. Restriction mapping this establishes a physical map of restriction sites sites where a particular restriction enzyme cuts on a fragment on dna. Lab 12 plasmid mapping student guide goal the objective of this lab is to perform restriction digestion of plasmid dna and construct a plasmid map using the results. To create a restriction map the unknown dna sample known as plasmid a by cutting it and separating it via agarose gel to. Store plasmid dna, restriction enzymes, 10x assay buffer and 1 kb. The unique restriction sites in plasmid help to insert the foreign dna into the plasmid. Restriction mapping an overview sciencedirect topics. Bio 6 restriction enzyme mapping lab objectives in this laboratory you will cut plasmid dna with various combinations of restriction enzymes to map the relative positions of the corresponding restriction sites on the plasmid. Pdf correlation of antibiotic resistance and restriction. Restriction map of plasmid prsetb 3973b part and a 2488b part. Long exposure to alkaline conditions may cause the plasmid dna to become irreversibly denatured. Dna technology mapping a plasmid a first step in working with dna is mapping the dna molecule. Ligase joins the fragment of foreign dna to the plasmid.
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. Restriction mapping requires the use of restriction enzymes. Snapgene viewer free software for plasmid mapping, primer. You digest all three plasmids puc, plasmid x, plasmid y with ecori, bamhi. Restriction endonuclease analysis rea of the mycoplasma genome provides a. The results of colony pcr came up with positive band at the right size, but after restriction mapping, it becomes clear that the insert is not in plasmid. We compared restriction enzyme analysis of plasmid reap dna profiling with bacteriophage typing for determination of similarities and differences among 50 pairs of. How to read a plasmid map features of a plasmid mapp.
The objective of this lab is to perform restriction digestion of plasmid dna and construct a plasmid map using the. Hiper restriction mapping teaching kit himedia laboratories. Physical map, restriction mapping problem definition. Restriction mapping in the molecular biology lab lawrence. The objective of this experiment module is to develop an understanding of the principles of dna mapping using various restriction enzymes to generate. Objectives after completion, the student should be able to. Using restriction mapping to teach basic skills in the molecular. This dna will run faster on a gel and is resistant to restriction enzyme digestion. Correlation of antibiotic resistance and restriction mapping of plasmid dna isolated from e. Although the newcomer likely knows that a plasmid is a small circular piece of dna found in bacterial cells, she may need some extra guidance to understand the specific components that make up a plasmid and why each is important. Our unique pglo plasmid map is available for educational use only.
How i tricked my brain to like doing hard things dopamine detox duration. Digested with hpai, hpaipsti, hpaisspi, and hpaipstisspilambda dna hpai. Two restriction enzymes, a and b, were used to obtain two individual digests, a and b. Jun 24, 2014 a restriction map is a map of known restriction sites within a sequence of dna. Because a 2488b chunk was cut by scai and bamhi in lane 5, the obvious restriction site for psti was very close to one of the bamhi sites. Dna fragments of this length are useful in the lab, since they long enough to contain the coding sequence for proteins and are wellresolved on agarose gels. Restriction mapping is a physical mapping technique which is used to determine the relative location of restriction sites on a dna fragment to give a restriction map. Therefore, we can use the recognition sequence as a flag to mark a map.
A diagram of the resulting gel is shown in figure 7. When the plasmid dna digested with combinations of restriction enzymes and the resulting fragments are electrophoresed, the following data is obtained. Mapping will be accomplished by performing single and double restriction digests of the plasmid followed by separation of the dna fragments using agarose gel electrophoresis. When such a vector is treated with a restriction enzyme that recognizes a recognition sequence in the polylinker, it is cut at that sequence, generating sticky ends. Would you like to move beyond handdrawn plasmid maps. Intact supercoiled plasmid dna and dnas with a large number of the target restriction site require more units of enzyme two to tenfold per. You identify two different plasmids, plasmid x and plasmid y, that contain yfg inserted into puc. N denotes the set of nonnegative integers for a set x of points on the line, let. Foreign dna is mixed with the plasmid and the plasmid is treated with the enzyme ligase. Experiment 2 plasmid dna isolation, restriction digestion. Practice problem 2 an unknown plasmid is digested with ecori and hindiii, and the fragments are separated by. Restriction mapping of bacterial dna linkedin slideshare. The restriction mapping method is used for the mapping of genes on a plasmid or shorter piece of dna although mapping the entire genome using the restriction digestion or restriction method.
Restriction mapping has widely publicized applications. Results are analyzed using agarose gel electrophoresis and a standard curve. Below is a restriction map for the plasmid pgen101 total length 20 kb. The plasmid also contains some selectable markers or the markers may be inserted in order to confirm the transformation of the exogenous gene. Restriction maps a map showing positions of restriction sites in a dna sequence if dna sequence is known then construction of restriction map is a trivial exercise in early days of. Restriction mapping of plasmid a branden michaelis abstract. One way to do this is to use restriction enzymes restriction endonucleases that are. Physical mapping of genomes is an important part of modern molecular genetics. Restriction enzymes are endonucleases that recognize specific sequences on dna and make specific cuts. For example, the enzyme ecori cuts dna at the sequence gaattc. Restriction map of plasmid a with the bamhi, psti, and scai enzymes. Restriction mapping since restriction enzyme recognition sequences are specific, they should occur at specific locations on every, identical dna molecule. All reactions are run to completion and the resulting digests give the following results. The enzymes hydrolyze the phosphate backbone creating a nick in the dna strand.
Use this information to create a restriction map of the plasmid. Practice problem 2 an unknown plasmid is digested with ecori and hindiii, and the fragments are separated by gel electrophoresis. Bio 6 restriction enzyme mapping lab objectives in this laboratory you will cut plasmid dna with various combinations of restriction enzymes to map the relative positions of the. Watch the video below to learn how to analyze your restriction digest results. Student practice problems name211174 211175 daterestriction mapping of plasmid dnaproblem 1. Using this map as a guide, give the number of restriction fragments. In this exercise, you will digest the plasmid pbr322 with 5 different restriction enzymes and resolve the fragments by agarose gel electrophoresis. A restriction map of a plasmid showing cut sites for ecori and bamhi. In molecular biology, restriction maps are used as a reference to engineer plasmids or other relatively short pieces of dna, and sometimes for longer genomic dna. These enzymes are produced by bacteria to protect themselves from bacteriophage attack. Note the origin position as well as the reference molecular weight markers on the gel. Bacteria produce restriction enzymes as a defense against invading viral dna.
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