To be more tricky, give each student group dna evidence that leads to different perpetrators, i. Dna restriction analysis in this experiment, dna from the bacteriophage lambda 4 8,502 base pairs in length is cut with a variety of restriction enzymes and the resulting fragments are separated using gel electrophoresis. L uncut lambda dna p psti restriction digest of lambda dna. Restriction analyzer an online restriction analysis tool. Restriction enzyme cleavage of dna student guide answers keywords. Dna restriction analysis is at the heart of recombinant dna technology and of the laboratories in. Students estimate the size of the fragments and determine which restriction enzyme was used to digest the dna. A restriction enzyme, is an enzyme that cuts double stranded dna following its specific recognition of short nucleotide sequences, is used to cut the dna into small fragments. The ecori restriction endonuclease with bacteriophage. Specifically, the restriction sites were mapped as follows.
The unique recognition sequences are usually tetra or hexanucleotide palindromes with axes of dyad symmetry. Under certain conditions of ph and ionic strength, the rates for the cleavage of the dna were the same at each recognition site. Restriction enzyme analysis of dna see page 3 for storage instructions. Run gels of digested dna restriction analysis of lambda carolina biological company kit. The case of the crown jewels is an activity that simulates the dna fingerprinting process used by forensic scientists, which relies on restriction analysis to analyze dna evidence from a. Compared to linear dna, intact supercoiled plasmid dna and dnas with a large number of the target restriction site require more units of enzyme. The size of these fragments is measured in base pairs or kilobase bases pairs. Many restriction enzymes have the ability to recognize a 4 to 10 nucleotide base pair palindrome. Dna digested with different restriction enzymes will serve as a. The ecori restriction endonuclease with bacteriophage lambda.
Restriction enzyme digestion lab report restriction. Introduction special enzymes termed restriction enzymes have been discovered in many different bacteria and other singlecelled organisms. A circular bacterial plasmid pbp1 has a single hindiii restrictionenzyme site in the. To investigate the efficiency and outcome of cutting singledigested lambda dna with the restriction enzyme ecori, using wealtecs cb1 block cooler as incubation system. This video demonstrates how to set up a restriction digest using the biorad restriction digestion and analysis of lambda dna kit. These restriction enzymes are able to scan along a length of dna looking for a particular sequence of bases that they. Restriction enzyme cleavage of dna student guide answers author. The case of the crown jewels is an activity that simulates the dna fingerprinting process used by forensic scientists, which relies on restriction analysis to analyze dna evidence from a fictional crime scene. These enzymes give us the ability to cut dna in a highly specific and reproducible way. Setting up a restriction enzyme digestion an analyticalscale restriction enzyme digestion is usually performed in a volume of 20. Gel electrophoresis will be employed to separate the resulting dna fragments. Place the dried gel overnight between two heavy books to avoid curling.
In this experiment, restriction enzymes are introduced as a tool to digest dna at specific nucleotide sequences. Learn vocabulary, terms, and more with flashcards, games, and other study tools. State how this affects electrophoresis of the uncut and cut dna. Application note l01 restriction digestion of lambda dna. Restriction enzyme tools are available on the web and on our mobile apps.
Restriction digestion and analysis of lambda dna kit. In this experiment, a full restriction digestion was performed. Dna restriction analysis is a technique with wide ranging applications in medicine, research, and forensics. Most restriction enzyme reactions are incubated at 37c for one hour. These sites are determined by the sequence of bases which usually form palindromes. Restriction analysis definition of restriction analysis. These differences in migration rate cause the fragments of. An example of gel electrophoresis of the fragments resulting from the sma1 restriction enzyme digestion modeled above would look like the illustration in fig. Palindromes are groups of letters that read the same in both the forward and backwards orientation. There may be a situation where the restriction enzyme has higher activity or the dnaase has lower activity.
Once the restriction enzyme cuts the dna, it creates either a blunt or sticky end. In the next procedure, you will see how to analyze separate dna. Restriction digests are mixtures of dna fragments produced by the reaction of dna and a restriction enzyme, an enzyme that cuts at specific base sequences. Restriction enzyme digestion lab report restriction enzyme. Edition, or the most recent version of the book, molecular biology and. A substance whose presence is essential for enzyme activity. Bary childrens science book award aps education center editorial board archive advanced. Biorad restriction digestion and analysis of lambda dna kit.
Perhaps, the ecori enzyme is more adept at splicing the lambda dna since it comes from a bacteria and thus when it acts upon a bacteriophage it is very effective. Separation by agarose gel electrophoresis of digests of lambda dna will yield separate bands corresponding to the dna fragments. A fragment of human genomic dna for gene p is excised from a. This type of wrk is vital for scientific research since dna splicing allows for various other genetic. This lesson focuses on restriction enzymes and their applications to dna analysis and dna fingerprinting. Over 100 fragments from single, multiple, and partial enzyme digestions were measured versus standards that were calibrated with respect to dna molecules of known sequence. Restriction enzyme cleavage of dna student guide answers. Lambda dna is the dna of a bacteriophage as seen below, and it attacks the human body by inserting its nucleic acid into the nucleus of the host cell.
To investigate the efficiency and outcome of cutting singledigested lambdadna with the restriction enzyme ecori, using wealtecs cb1 block cooler as incubation system. These enzymes are used for the creation of genetic maps prior to sequencing. Wards analyzing dna with restriction enzymes lab activity. Three samples of purified dna from bacteriophage 48,502 bp in length are incubated at 37c, each with one of three restriction endonucleases. In the next procedure, you will see how to analyze separate dna fragments with gel electrophoresis. Restriction digestion and analysis of lambda dna kit life science. A was added to eppendorf tubes to a final volume of 20. This online tool helps you analyze restriction enzyme cutting maps. The unit is the standard measure of restriction enzyme activity and is defined as the amount of enzyme needed to digest to completion 1 l if dna in a 50 l reaction in 1 hour. The discovery of restriction enzymes and their applications in dna analysis has proven to be essential for biologists and chemists. The restriction digestion took place overnight and was kept in the freezer until the next class period when it was used for gel electrophoresis.
If an unusually large volume of dna or enzyme is used, aberrant. Restriction enzyme mapping is a powerful tool for the analysis of. Twosession laboratory activity, 45 min per session. If an unusually large volume of dna or enzyme is used, aberrant results may occur. This, in turn, has ushered in the area of molecular cloning, mapping and sequencing the fine genetic structure of dna. This is a paper exercise where you will use images of restriction enzyme digestion data to construct a physical map of the phage lambda genome. Education center k12 lessons and laboratories classroom activities in plant biotechnology. The pattern of restriction fragments produced can be used for various purposes. In this investigation, students observe the effects of three restriction enzymes on lambda. These restriction enzymes are able to scan along a length of dna looking for.
Gel electrophoresis of precut lambda dna, precut plasmid dna. Feb 15, 2016 this occurred because the enzymes broke apart the lambda dna in different ways. Restriction enzyme resource guide promega corporation. You can purchase samples of lambda dna cut with other restriction enzymes from commercial vendors, or you can combine a sample of lambda dna cut with ecori with a sample cut with hindiii to mix things up. Use this lesson and its associated activity in conjunction with biology lessons on dna analysis and dna replication. After incubation, you can analyze the dna or use it in other kinds of reactions, such as the bacterial transformation you did in the first part of this lab. A map of bacteriophage lambda was constructed, including accurate positions for all 41 cut sites made by 12 different restriction enzymes.
Restriction digestion and analysis of lambda dna kit aligns with ap biology lab six. Genscript restriction enzyme map analysis tools help you analyze restriction enzyme cutting maps. The discovery of restriction enzymes has ushered in a new era of molecular genetics. Select from an extensive catalog of educational tools available for enhancing the teaching of biological subjects, including simulated testing of blood and urine, the illustration of various natural habitats, and the dissection of sterilized stool samples collected from different regions. Freebies lesson and lab exchange high schoolcollege dna. Restriction digestion and analysis of lambda dna students use three different restriction enzymes to digest genomic dna from lambda bacteriophage. Restriction analysis of lambda dna terrific science. Restriction enzyme analysis of dna san pedro high school.
The objective of this experiment is to develop a basic understanding of the role of restriction enzymes and agarose gel electrophoresis to cut and size dna. Oct 16, 2012 this video demonstrates how to set up a restriction digest using the biorad restriction digestion and analysis of lambda dna kit. Manipulation of restriction targets in phage lambda to form receptor chromosomes for dna fragments. A restriction enzyme map is a basic requirement for the detailed structural and functional analysis of genes. The gels were stained overnight prior to photographing or recording results. With the restriction digestion and analysis of lambda dna kit, students use three different restriction enzymes to digest genomic dna from lambda bacteriophage. Analysis of eco ri cleavage patterns of lambda dna. Variations in cleavage patterns obtained from lambda. Plus, get practice tests, quizzes, and personalized coaching to help you succeed.
By visualizing the effects of three different enzymes on identical samples of doublestranded dna, students learn that different restriction enzymes recognize and cut different dna sequences. What is the goal of the lambda dna restriction analysis lab. While it is easy to illustrate restriction mapping with dry paper and pencil examples, suitable handson wet practical experiments are more difficult to devise. Theses newly cut molecules are called dna fragments. By using the same restriction enzyme to cut dna from two. Separation by agarose gel electrophoresis of an eco ri digest of lambda dna will yield 6 bands 5 distinct bands, two are very close in size corresponding to the dna fragments. Restriction enzyme analysis of dnastudent laboratory kit. Label four micro test tubes l, p, e, and h and place them in the foam microtest tube holder. Variations in cleavage patterns obtained from lambda dna digested. Electrophoretic techniques that distinguish dna fragments by size are essential in forensics and in the mapping of restriction sites within genes. Restriction enzyme mapping of the lambda phage genome.
This protocol introduces the analysis of dna using restriction enzymes. Restriction enzymes were a catalyst for the molecular biology revolution, and now hundreds of such enzymes are known. The groups stop the restriction digests at appropriate times extending over one hour by addition of 1 i1 of 0. As a member, youll also get unlimited access to over 79,000 lessons in math, english, science, history, and more. Scientists discovered restriction enzymes in 1962 while studying bacteria that appeared to possess a primitive immune system that prevented viral dna from replicating within the infected host bacterium. Because the number of ddei recognition sites may be different for each student, agarose gel electrophoresis is then implemented to detect the different digest patterns or haplotypes found in the class. The restriction enzyme will continue to do this along the full length of the dna molecule which will then break into fragments. By agreement with the publisher, this book is accessible by the search feature, but. The restriction enzyme ddei is then used to perform a restriction analysis of the dna. The restriction enzyme ecori cuts dna at the sequence gttaac, and the. Nov 01, 1980 the kinetics of the reactions of the ecori restriction endonuclease at individual recognition sites on the dna from bacteriophage lambda were found to differ markedly from site to site.
Restriction enzyme lab report essay example graduateway. The dna is cleaved with the enzymes and the fragments obtained are generally separated by gel electrophoresis. Restriction endonucleases in the analysis and restructuring of dna molecules. Restriction endonucleases, or restriction enzymes, are important classes of enzymes used by biotechnologists.
Lambda dna is a linear dna form that is an industry standard for measuring and expressing unit activity for many restriction enzymes. Dna digested with different restriction enzymes will. These differences in migration rate cause the fragments of dna to separate from one another over time. Each student lab group can be responsible for casting their. If you dont know why you are doing something, ask each other and then your teacher for clarification.
Bacteriophage lambda dna has a linear structure and multiple recognition sites. In this investigation, the restriction enzymes ecori, psti, and hindiii will be used to digest bacteriophage lambda dna. Obtain micro test tubes that contain each enzyme solution, lambda dna, and restriction buffer from the common station. The kinetics of the reactions of the ecori restriction endonuclease at individual recognition sites on the dna from bacteriophage lambda were found to differ markedly from site to site. Restriction enzymes are endonucleases that cleave both strands of dna at. Restriction analyzer provides comprehensive restriction analysis of a dna molecule. Restriction enzyme activity is adjusted by the instructor prior to the exercise such that diluted enzyme will digest 50% of the plasmid dna in 540 min. Plasmid is circular dna 4,500 base pairs, lambda is linear dna 49,000 base pairs. If digests are not complete, try doubling the amount of enzyme you add to each reaction. Restriction enzyme analysis methylene blue stain student activity restriction enzymes cut at specific sites along the dna. Bateriophage lambda dna has a linear structure and 6 eco ri recognition sites. Hind iii digestion of lambda dna, may not be visible after separation on.26 1484 356 378 980 989 183 1553 162 917 801 987 1277 1003 909 739 719 824 349 259 1208 371 1199 688 1022 1466 18 1141 571 1364 162 215 591 1060 952 734