Multiple plasmid constructs can be analyzed simultaneously for the presence or absence of an insert, orientation of the insert, plasmid size, and some sitespecific sequence data. Place the cracker pieces in separate squares of waxed paper. As they cut within the molecule, they are commonly called restriction endonucleases. Spit the cracker and saliva mix into the second bowl.
Restriction enzyme digestion lab report restriction. Restriction digestion procedure sakshat amrita virtual lab. This lab will help learners understand what a dna restriction enzyme is and how it works, how to use a micropipette, how to separate dna using electrophoresis, and how to use a restriction digestion map to identify a sample dna. Restriction enzyme digestions are performed by incubating doublestranded dna molecules with an appropriate amount of restriction enzyme, in its respective buffer as recommended and often supplied by the commercial supplier, and at the optimal temperature for that specific enzyme. These restriction endonucleases make a cut wherever that sequence occurs in the dna. Restriction enzyme digestion lab report restriction enzyme. Molecular biology protocol restriction digest of plasmid.
A restriction digest is a procedure used in molecular biology to prepare dna for analysis or other processing. It is also used to quickly check the identity of a plasmid by diagnostic digest. Multiple plasmid constructs can be analyzed simultaneously for the presence or absence of an insert, orientation of the insert. When the cracker is in your mouth, your saliva starts to break the bonds holding the cracker together as well as mix with it. The insert size and orientation was examined by estimating the fragment sizes using a reference standard. Some work at lower temperatures 25c, sma 1 while taq i works at 65c. The restriction digestion took place overnight and was kept in the freezer until the next class period when it was used for gel electrophoresis. A partial restriction digest involves performing an incomplete digestion of the plasmid dna so that, in our example where you have two restriction sites for the enzyme in question, you will end up with three digestion products. Three samples of lambda p hage dna are incubated at 37 degrees c. This is particularly true if you receive a plasmid from someone in another lab.
Which restriction enzyme should be used to isolate the lactase gene from dna. May 18, 2018 a diagnostic restriction enzyme digest takes advantage of the fact that restriction enzymes cleave dna at specific sequences called restrictions sites. Since a restriction enzyme may lose activity due to improper storage or handling, it is important that you always. I am now conducting a restriction enzyme digestion using smai, which will cut the dna at only a single site. Restriction enzyme digestion principle shomus biology. Digest, chew, swallow, saliva, stomach, acid, intestine, elimination. Restriction enzyme lab by courtneyann taylor on prezi. Instruct the students to choose a fresh cracker and chew it, no swallowing allowed. A given restriction enzyme cuts dna segments within a specific nucleotide sequence, at what is called a restriction site. Experiment 6 lab periods 5 and 6 restriction enzymes and. I conducted a pcr reaction using a total of 2 ul of 10mm dntps.
A digestion reaction typically consists of the following. Most restriction enzymes function optimally at restriction enzymes are used to produce a pool of dna fragments to be cloned. 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. A quik way around partial restriction digests bitesize bio. The gel is then stained with a methylene blue stain to visualize the dna bands and may be photographed.
Setting up a restriction enzyme digestion an analyticalscale restriction enzyme digestion is usually performed in a volume of 20. Typical incubation time and temperature is 37c for 1 hour, though time and temperature will vary depending on restriction enzyme used. Experiment 2 plasmid dna isolation, restriction digestion. Our labs protocols state simply to allow the digestion to run overnight.
If an unusually large volume of dna or enzyme is used, aberrant results. This package will allow you to work with restriction enzymes and realise restriction analysis on your sequence. The stomach model lab can be used to introduce the stomach or mechanical and chemical digestion, as well as provide students a chance to continue learning after the concepts have been introduced. Place the other half of the saltine cracker in the mouth and hold for 3 minutes without chewing or swallowing and record observations.
Would the restriction enzymes we used in lab be able to cut other types of dna besides the. Restriction digestion is accomplished by incubation of the target dna molecule with restriction enzymes enzymes that recognize and bind specific dna sequences and cleave at specific. Bio 6 restriction enzyme digestion lab objectives upon completion of this laboratory you will understand how to. Add 1 or 2 tablespoons of water to the cracker crumbs. Jan 07, 2020 write down in alphabetical order a list of the restriction enzymes you have available to you this could be your personal stocks, lab stocks, or those from another lab if youre on friendly enough terms. Restriction enzyme analysis of dna experiment objective. Identify any potentially mischievous restriction enzymes and try to avoid them i put an asterisk next to them. Restriction endonucleases cleave double stranded dna internally, acting essentially like a pair of scissors to cut both strands of the molecule. Digestion experiment weird unsocialized homeschoolers. Make a digestive system model using crackers and bananas.
Restriction digestion also called restriction endonuclease is a process in which dna is cut at specific sites, dictated by the surrounding dna sequence. 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. You must collect at least 5 ml one teaspoon of saliva. Restriction enzymes restriction endonucleases are proteins that cut dna at or close to specific recognition sites see the catalogs of manufacturers or the. Chew and swallow half of the saltine cracker and record observations on the texture and flavor of the cracker 3. These special enzymes recognize specific sequences in the dna molecule for example gatatc wherever that sequence occurs in the dna. Choosing restriction enzymes whose recognition sequences flank your gene of interest.
Is the purpose of partial restriction enzyme digest to reduce length of. Students will be exposed to the major steps of digestion. Duplication of any part of this document is permitted for classroom. Experiment 2 plasmid dna isolation, restriction digestion and gel electrophoresis plasmid dna isolation introduction. The application of molecular biology techniques to the analysis of complex genomes depends on the ability to prepare pure plasmid dna. This was done using alkaline lysis method of plasmid dna isolation followed by restriction digest and gel elecrophoresis of it.
If your class advanced, you can mention that breaking something into smaller pieces also increases the surface area, therefore, makes chemical digestion more efficient. In such cases make sure that, it is added to the reaction mixture. This makes the cracker softer and easier to swallow. I wanted to know if there was a more defined time needed for a complete reaction, as id like. Free stomach model digestion lab by science teacher. Restriction enzymes digestionrestriction endonuclease. You can take this experiment further and add a few squirts of different coloured food colouring to the food to mimic some of the digestive juices. Restriction enzymes are nucleases which can cleave the sugarphosphate backbone of dna, found in bacteria. Materials per partner group three crackers and two petri dishesplates one small bottle of iodine and dropper pencil procedure. A restriction enzyme or restriction endonuclease recognizes a specific nucleotidepair sequence in dna called a restriction site and cleaves the dna hydrolyzes the. Nucleic acid techniques, molecular biology, center for cardiovascular research. Salts like nacl or mgcl2 have to be added to serve as a buffer and maintain. They specifically cleave the nucleic acids at specific nucleotide sequence called restriction sites to generate a set of smaller fragments. Restriction digestion and analysis of lambda dna kit.
Experiment 2 plasmid dna isolation, restriction digestion and. Data hypothesis restriction enzyme lab if we use different enzymes to cut dna, then, hindiiii will be the most effective because it will cut the dna at a distance of 23,bp which. Jan 29, 2010 nucleic acid techniques, molecular biology, center for cardiovascular research. Guess i am spoiled by the convenience of having a neb cabinet here right next to our lab. Place five drops of iodine on the cracker in the petri dish. Learn enzyme digestion restriction with free interactive flashcards. Prepare positive control reaction with template of known cutting site corresponding to the restriction enzyme of choice. A diagnostic restriction enzyme digest takes advantage of the fact that restriction. This experiment is designed to develop an understanding of restriction enzyme digestion and separation of dna fragments using agarose gel electrophoresis. Students will be able to model and describe mechanical and.
Learn vocabulary, terms, and more with flashcards, games, and other study tools. An enzyme called salivary amylase begins to catalyze the breakup of starches by hydrolysis. Demonstrate how each part of the digestive system works using crackers, pantyhose, create teeth molds, prepare and eat a salad while discussing healthy eating habits, and more. A restriction enzyme digest should be carefully planned. Often, the size of the plasmid insert and vector backbone are known and thus this technique can be quickly used to verify your plasmid. Preparation of dna for traditional cloning methods is dependent upon restriction enzyme digestion to generate compatible ends capable of being ligated together.
This restriction enzyme troubleshooting guide organizes solutions to common issues encountered when performing digestions with restriction endonucleases. Chewed not chewed texture flavor texture flavor analysis questions. Restriction enzyme digestion is commonly used in molecular cloning techniques, such as pcr or restriction cloning. And comparing them with restriction maps of all possible insert configurations. The digestion activity of restriction enzymes depends on the following factors. Restriction enzymes thermo scientific tae buffer trisacetateedta 50x optimize electrophoresis of both genomic and large supercoiled dna in agarose and polyacrylamide gels with thermo scientific 50x tae buffer trisacetateedta. Understanding the concepts of molecular biology and then applying those concepts to laboratory experiments can be challenging to entrylevel students. Watch the video below to learn how to analyze your restriction digest results. Restriction enzyme digestion principle to analyze genomic dna, we must first cut it into smaller, more manageable pieces. Incubating the reaction for the recommended amount of time. The salivary glands secrete saliva, an alkaline fluid, to soften food, moisten the mouth and aid in the swallowing process. Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave dna at specific sequences. In this experiment, we will use restriction enzymes to cut up dna from a small virus called bacteriophage this virus is 48,502 base pairs in length which is very.
Write down in alphabetical order a list of the restriction enzymes you have available to you this could be your personal stocks, lab stocks, or those from another lab if youre on friendly enough terms. Restriction enzymes are typically inactivated by incubation at high temperature. Please help me with calculations for restriction enzyme digestion experiment. In order to facilitate the topics of restriction enzyme digestion and the generation of compatible ends in the process of gene cloning, an inclass activity was designed. These recognition sequences are typically four, six, eight, ten, or twelve nucleotides long and generally palindromic i. Restriction enzyme digestion and ligation thermo fisher. Restriction enzyme digestion exercise an inclass activity. It is sometimes termed dna fragmentation this term is used for other procedures as well. Cloning cloning methods cloning using restriction enzymes embl. Add one tablespoon of water, and then add two drops of tincture of iodine. Restriction make use of the facilities offered by rebase and contains classes for more than 800 restriction enzymes. Restriction digestion theory sakshat amrita virtual lab. Introduction to the myth heres how it goes give students saltine crackers. Starch digestion in your mouth vancleaves science fun.
When you are done please check out human digestion demonstration part 2 the bread in the bag. Is the purpose of partial restriction enzyme digest to reduce length of experiment or to see which restriction enzyme cuts the largest dna. Please help me with calculations for restriction enzyme digestion. Restriction digestion of recombinant plasmid constructs provides a fast, costefficient method of gaining indirect sequence information. Chew the other half of the saltine cracker or 2 or 3 oyster crackers in your mouth very well so that it mixes with the saliva. Restriction enzymes, found naturally in bacteria, can be used to cut dna fragment at specific sequences. 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 phosphates and hydroxyls. This simulation provides an opportunity to practice a restriction digest in a virtual lab setting.
After performing the experiment, confirm the digestion of dna by running a small amount of it in agarose gel with an undigested standard dna. This enzymatic technique can be used for cleaving dna molecules at specific sites, ensuring that all dna fragments that contain a particular sequence at a. Protocol how to perform a diagnostic digest addgene. Digestion by a restriction enzyme generates dna fragments with one of two types of dna endssticky or blunt. To perform restriction digestion of dna with ecor i and bamhi enzymes. Bsa will stabilize the reaction by preventing enzyme from sticking to the. Restriction enzyme troubleshooting guide thermo fisher scientific. A diagnostic restriction enzyme digest takes advantage of the fact that restriction enzymes cleave dna at specific sequences called restrictions sites. The act of chewing is the mechanical digestion as chewing helps to break the cracker into smaller pieces so that its easier to swallow. Most plasmid dna isolation techniques come in two flavors, simple low quality dna preparations.
Break off two pieces of cracker approximately the same size about the size of a small pea. The dna to be cloned can vary widely, from genomic dna extracted from a pure bacterial culture or a mixed population, to a previously cloned gene that needs to be moved from one vector. Digestive system lesson this is part 4 of a 7 part handson unit study on anatomy of the human body. Lab periods 5 and 6 restriction enzymes and restriction fragments restriction enzymes or restriction endonucleases are bacterial proteins that act as defense mechanisms in these organisms. P psti restriction digest of lambda dna violet tube e ecori restriction digest of lambda dna green tube h hindiii restriction digest of lambda dna orange tube 2. Identify any potentially mischievous restriction enzymes and. Choose from 500 different sets of enzyme digestion restriction flashcards on quizlet. Restriction digestion is the process of cutting dna molecules into smaller pieces with special enzymes called restriction endonucleases sometimes just called restriction enzymes or res. The process of restriction digestion is carried out by mixing the dna and the restriction enzymes together. The ability to cleave dna at specific sites is one of the cornerstones of todays methods of dna manipulation. The product is exactly 100 bp, and 35 cycles were run. Most endonucleases digest the target dna at 37c with few exceptions. View lab report restriction enzyme digestion lab report from biol 211 at claflin university.
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