Restriction Enzyme Lab Report Essay Example | Graduateway

 

restriction enzyme digestion lab report

View Lab Report - Restriction Enzyme Digestion Lab Report from BIOL at Claflin University. Restriction Enzyme Digestion Lab Report Tranard J. Harvin Lab Partners: Whyeisha Jones and Allison82%(33). Oct 02,  · Restriction Digestion of Plasmid DNA using Agarose Gel Electrophoresis - Free download as Word Doc .doc), PDF File .pdf), Text File .txt) or read online for free. Report on Restriction Digestion of Plasmid DNA using Agarose Gel Electrophoresis for /5(12). Restriction Digestion and Analysis of Lambda DNA Kit Instruction Manual Catalog #EDU voliogdasa.gq The kit is packaged and shipped as two modules. Open the modules immediately upon receipt and store components at –20°C, 4°C, or room temperature as indicated. Duplication of any part of this document is permitted for classroom.


dna restriction analysis lab report | Gel Electrophoresis | Restriction Enzyme


Special enzymes termed restriction enzymes have been discovered in many different bacteria and other single-celled organisms. These restriction enzymes are able to scan along a length of DNA looking for a particular sequence of bases that they recognize. This recognition site or sequence is generally from 4 to 6 base pairs in length. Once it is located, the enzyme will attach to the DNA molecule and cut each strand of the double helix.

The restriction enzyme will continue to do this along the full length of the DNA molecule which will then break into fragments. The size of these fragments is measured in base pairs or kilobase bases pairs. Since the recognition site or sequence of base pairs is known for each restriction enzyme, we can use this to form a detailed analysis of the sequence of bases in specific regions of the DNA in which we are interested. In the presence of specific DNA repair enzymesDNA fragments will reanneal or stick themselves to other fragments with cut ends that are complimentary to their own end sequence, restriction enzyme digestion lab report.

This DNA may contain genes that allow the organism to exhibit a new function or process. This would include transferring genes that will result in a change in the nutritional quality of a crop or perhaps allow a plant to grow in a region that is colder than its usual preferred area.

This virus is 48, base pairs in length which is very small compared with the human genome of approximately 3 billion base pairs. If the virus DNA is exposed to the restriction enzyme for only a short time, then not every restriction site will be cut by the enzyme. This will result in fragments ranging in size from the smallest possible all sites are cut to in-between lengths some of the sites are cut to the longest no sites are cut. This is termed a partial restriction digestion.

In this experiment, we will perform a full restriction digestion. After overnight digestion, the reaction is stopped by addition of a loading buffer. The DNA fragments are separated by electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate into an agarose gel.

The gel is then stained with a methylene blue stain to visualize the DNA bands and may be photographed. This laboratory will take approximately 3 days. The restriction digestion takes place overnight and can be kept in the freezer until the next class period when it will be be used for gel electrophoresis. The gels may be stained overnight prior to photographing or recording results.

Gels may be discarded in regular trash receptacle. A description of how to use a micropipet can be found in Activity 2 - Gel Electrophoresis of Dyes. Although methylene blue dye is not as sensitive as ethidium bromide it may be used to stain the higher quantities of DNA that are used in this experiment. Methylene blue is non-toxic but will stain clothes, hands, and equipment, so always wear gloves. Use the stain close to a sink and clean up spills immediately.

Use distilled or deionized water to de-stain gels. Only use deionized water for making the 0. A single container of methylene blue dye should be all that is needed since it may be reused several times and disposed of restriction enzyme digestion lab report the sink.

Restriction enzyme digestion lab report enzymes require special care for handling and use. They lose activity unless kept frozen; exposure to warm temperatures for even a short time will result in loss of activity.

Using good sterile technique, aliquot samples for students, being careful to keep everything on ice until ready to be used. Enzymes should be stored in a foam container in the freezer non frost-free if availablealong with the special buffer for each enzyme. The special buffers contain the salt and pH requirements for optimal activity of each enzyme. Since viruses have a relatively simple genome, scientists have studied their DNA and used this information to test theories and develop concepts that apply to the genetics of living organisms.

The information from the relatively simple virus genomes has been used to test theories and develop concepts that apply to the genetics of living organisms, restriction enzyme digestion lab report.

Each enzyme recognizes a unique sequence of bases along the DNA strand and cuts the strand at these sites - the first step in a process called restriction mapping. This procedure is one of the most important in modern biology. The small fragments of DNA are separated by gel electrophoresis. The movement of the fragments will always be towards the positive electrode because DNA is a negatively charged molecule.

The fragments move through the gel at a rate that is determined by their restriction enzyme digestion lab report and shape, with the smallest moving the fastest, restriction enzyme digestion lab report.

DNA cannot be seen as it moves through the gel. A loading dye must be added to each of the samples before it is pipetted into the wells. The progress of the dye can be seen in the gel. It will initially appear as a blue band, eventually resolving into two bands of different colors.

The faster moving, purplish band is bromophenol blue dye that migrates at roughly the same rate as a base pair fragment of DNA. The slower moving aqua band is xylene cyanol, restriction enzyme digestion lab report, nearly equivalent to a base pair fragment.

The faster moving band must move at least cm from the wells to achieve the best separation of DNA for analysis. Care should be taken not to let the bromophenol blue band run off the end of the gel.

Following staining to locate the DNA, the gel is observed and the fragments appear as a pattern of bands. In this experiment, we will compare our banding pattern with a predicted result shown in figure 1. Information may be provided by your teacher that details the process of isolating and analyzing these bands to create a DNA fingerprint. The methylene blue dye will stain skin, clothes, and equipment. Always wear gloves and safety glasses.

Do all staining in a central area near the sink. Restriction enzymes cut at specific sites along the DNA. These sites are determined by the sequence of bases restriction enzyme digestion lab report usually form palindromes.

Palindromes are groups of letters that read the same in both the forward and backwards orientation. The complimentary bases on the opposite strand will be CTTAAG, which is the same as reading the first strand backwards!

Many enzymes recognize these types of sequences and will attach to the DNA at this site and then cut the strand between two of the bases.

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Activity 3: Restriction Enzyme digestion - How does it work? Why is it useful? Page Content. Introduction Special enzymes termed restriction enzymes have been discovered in many different bacteria and other single-celled organisms. Objectives Understand what a DNA restriction enzyme is and how it works. Learn to use a micropipette. Learn to separate DNA on an agarose gel using electrophoresis.

Understand how to use a restriction digestion map to identify a sample DNA. Obtain enough crushed ice and ice containers styrofoam cups for each lab group.

Reconstitute the lambda DNA with sterile distilled water to 0. Aliquot lambda DNA, enzymes and loading dye for each group and keep in freezer until needed. Make the restriction enzyme digestion lab report. Heat in the microwave for 30 seconds at a time, shaking gently restriction enzyme digestion lab report time, until the agarose is completely melted.

Alternatively, the solution can be heated on a hot plate, with occasional gentle shaking, until the agarose is melted. Keep warm if the class will use it within a half hour, restriction enzyme digestion lab report. Otherwise, allow the solution to cool and solidify. Cover and keep in the refrigerator. Pour enough agarose gels for each lab group as follows: Wear gloves Microwave or warm the agarose bottle in a hot waterbath until the gel liquefies.

Be sure to use a microwave designated for science purposes not food. Firmly seal the ends of the gel tray using labeling tape. Place the plastic comb in the slots close to the end of the tray.

Pour approximately ml of agarose into each gel tray. Let cool until solidified approximately 15 minutes, restriction enzyme digestion lab report. If storing overnight, place trays in a container or ziploc baggie with 0. Day 3: Remove student gels from the refrigerator. Set up containers for staining in a common area near a sink, restriction enzyme digestion lab report.

Note Gels may be discarded in regular trash receptacle. Use of Methylene Blue: Although methylene blue dye is not as sensitive restriction enzyme digestion lab report ethidium bromide it may be used to stain the higher quantities of DNA that are used in this experiment. Background Reading Since viruses have a relatively simple genome, scientists have studied their DNA and used this information to test theories and develop concepts that apply to the genetics restriction enzyme digestion lab report living organisms.

Figure 1. Leach Laboratory Information may be provided by your restriction enzyme digestion lab report that details the process of isolating restriction enzyme digestion lab report analyzing these bands to create a DNA fingerprint.

Procedure Put on gloves. Keep all enzyme and DNA aliquots on ice through step 6. When adding the droplets of buffer to the restriction tube, touch the pipette tip to the bottom of the tube. Use a new tip for each buffer. Set the micropipette to 4.

 

Biochemistry 3 Example Report

 

restriction enzyme digestion lab report

 

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. Most plasmid DNA isolation techniques come in two flavors, simple - low quality DNA preparations. Restriction enzymes are enzymes (proteins that act as molecular tools) which cut DNA. Each restriction enzyme cuts DNA at a particular nucleotide sequence, acting like a molecular scissors. For instance, one enzyme that you will use in this lab, EcoRI, cuts DNA at the sequence 5’ -.G ATC 3’. This sequence of DNA is called the recognition. DNA Restriction. Analysis Lab. Rylee Kopchak Biology Period 8 May 14, Introduction In this experiment, the scientists used restriction enzymes to cut DNA from the bacteriophage lambda into fragments, which were then separated using gel electrophoresis. Restriction enzymes, also known as restriction endonucleases, cut a DNA molecule at a particular sequence known as the recognition site.5/5(7).