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Activity 3_ Restriction Enzyme Analysis

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  Share  Activity 3: Restriction Enzyme digestion - Howdoes it work? Why is it useful? Introduction Special enzymes termed restriction enzymes  have been discovered in manydifferent bacteria and other single-celled organisms. These restriction enzymesare 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 4to 6 base pairs in length. Once it is located, the enzyme will attach to the DNAmolecule and cut each strand of the double helix. The restriction enzyme willcontinue to do this along the full length of the DNA molecule which will thenbreak into fragments. The size of these fragments is measured in base pairs or kilobase (1000 bases) pairs.Since the recognition site or sequence of base pairs is known for each restrictionenzyme, we can use this to form a detailed analysis of the sequence of bases inspecific regions of the DNA in which we are interested.In the presence of specific DNA repair enzymes , DNA fragments will reannealor stick themselves to other fragments with cut ends that are complimentary totheir own end sequence. It doesn’t matter if the fragment that matches the cut end comes from the same organism or from a different one. This ability of DNAto repair itself has been utilized by scientists to introduce foreign DNA into anorganism. This DNA may contain genes that allow the organism to exhibit a newfunction or process. This would include transferring genes that will result in achange in the nutritional quality of a crop or perhaps allow a plant to grow in aregion that is colder than its usual preferred area.In this experiment, we will use restriction enzymes to cut up DNA from a smallvirus called Bacteriophage λ. This virus is 48,502 base pairs in length which isvery small compared with the human genome of approximately 3 billion basepairs. Since the whole sequence of λ is already known we can predict whereeach restriction enzyme will cut and thus the expected size of the fragments thatwill be produced. If the virus DNA is exposed to the restriction enzyme for only ashort time, then not every restriction site will be cut by the enzyme. This willresult in fragments ranging in size from the smallest possible (all sites are cut) toin-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 overnightdigestion, the reaction is stopped by addition of a loading buffer. The DNAfragments are separated by electrophoresis, a process that involves applicationof 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 bandsand may be photographed.This laboratory will take approximately 3 days. The restriction digestion takesplace overnight and can be kept in the freezer until the next class period when it Lessons andLabs Plant Biotech Activities HomeWhat is DNA?DiseasesHistoryHealthEnvironment ActivitiesConclusions  APS > Education > K-12 > Lessons and Laboratories > Classroom Activities in PlantBiotechnology > Activity 3: Restriction Enzyme Analysis  will be be used for gel electrophoresis. The gels may be stained overnight prior to photographing or recording results. Objectives 1. Understand what a DNA restriction enzyme is and how it works.2. Learn to use a micropipette.3. Learn to separate DNA on an agarose gel using electrophoresis.4. Understand how to use a restriction digestion map to identify a sampleDNA.5. Compare the λ DNA bands on a gel to the known λ DNA restriction map.  Materials For each lab group Four microtubesMicrotube rack20-µl micropipette (or 10-µl micropipette) and sterile tipsWaterproof penBeaker or foam cup with crushed ice for the following20 µl of 0.4 µg/µl λ DNA2.5 µl Bam HI restriction enzyme2.5 µl Eco RI restriction enzyme2.5 µl Hind  III restriction enzyme10 µl distilled water Gloves500-ml beaker (day 2)Electrophoresis chamber (day 2)Power supply (day 2)20 µl 10X loading dye (day 2)1.0% agarose gel (day 2) Common Materials Container with TBE solution (1X)37°C water bath w/ floating rack60°C water bath or saucepan on a hot plate (day 2)Cooler with crushed iceFreezer (non frost-free, if possible)Camera if desiredDistilled water 0.002% methylene blue stain (day 3)  Advance Preparation Day 1:  1. If you saved the 1X TBE solution from the Gel Electrophoresis with Dyesactivity, reuse it for this laboratory. 2. Obtain enough crushed ice and ice containers (styrofoam cups) for eachlab group. 3. Fill a pan with water and adjust it to 55°C on a hot plate 4. Fill a second pan with water and adjust it to 37°C on a hot plate while thestudents complete preparation of the restriction digests. 5. Reconstitute the lambda DNA with sterile distilled water to 0.4 µg/µl. 6. Aliquot lambda DNA, enzymes and loading dye for each group and keep infreezer until needed.  7. Make the 1.0% agarose gel solution as follows: To make 100 ml of gel, which is sufficient for 3 gels, weigh out 1.0 g of agarose and place into a 200- to 250-ml glass beaker or flask. Add 100 mlof 1X TBE (Tris-Borate-EDTA) buffer. Heat in the microwave for 30seconds at a time, shaking gently each time, until the agarose is completelymelted. Alternatively, the solution can be heated on a hot plate, withoccasional gentle shaking, until the agarose is melted. Keep warm if theclass will use it within a half hour. Otherwise, allow the solution to cool andsolidify. Cover and keep in the refrigerator. Day 2:  8. Fill a pan with water and adjust it to 60°C. 9. Pour enough agarose gels for each lab group as follows:Wear glovesMicrowave or warm the agarose bottle in a hot waterbath until thegel liquefies. Be sure to use a microwave designated for sciencepurposes (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 35-40 ml of agarose into each gel tray. Thiswill result in a thick gel so that at least 20 µl of sample can beloaded into each well.Let cool until solidified (approximately 15 minutes).If storing overnight, place trays in a container or ziploc baggie with0.5X TBE solution so they do not dry out. Day 3: 10. Remove student gels from the refrigerator. 11. Set up containers for staining in a common area near a sink. Note Gels may be discarded in regular trash receptacle. A description of how to use amicropipet can be found in  Activity 2 - Gel Electrophoresis of Dyes. Use of Methylene Blue:  Although methylene blue dye is not as sensitive as ethidium bromide it may beused 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, soalways wear gloves. Use the stain close to a sink and clean up spillsimmediately. Use distilled or deionized water to de-stain gels. Only use deionizedwater for making the 0.1X TBE buffer to make this stain since the high chlorinelevels of most tap water will damage the DNA. A single container of methyleneblue dye should be all that is needed since it may be reused several times anddisposed of down the sink. Use of Power Supplies See description in Gel Electrophoresis of Dyes -  Activity 2EnzymesRestriction enzymes require special care for handling and use. They lose activityunless kept frozen; exposure to warm temperatures for even a short time willresult 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 available), along with the special buffer for each enzyme. The special bufferscontain the salt and pH requirements for optimal activity of each enzyme. Lambda (λ) DNA: The λ DNA used in this laboratory can exist as either a linear or circular molecule, creating some confusion when interpreting restriction digest results. Byheating the sample to 60°C for 3 minutes, immediately prior to electrophoresis,the hydrogen bonds holding the ends of the linear DNA together in a circle will bebroken. Background Reading Since viruses have a relatively simple genome, scientists have studied their DNAand used this information to test theories and develop concepts that apply to thegenetics of living organisms. One of the most studied viruses is calledbacteriophage lambda (λ). Bacteriophage λ is a virus that infects bacterial cells. Student Activity: Restriction Enzyme Analysis - MethyleneBlue stain Background Reading Bacteriophage λ is a virus that attacks bacterial cells and is one of the moststudied viruses. The information from the relatively simple virus genomes hasbeen used to test theories and develop concepts that apply to the genetics of living organisms. The DNA of Bacteriophage λ is approximately 48,514 basepairs or 48.514 kilobase pairs in length while the human genome isapproximately 3 billion base pairs.This experiment uses special “restriction” enzymes that act as chemical scissorsto cut λ DNA into pieces. Each enzyme recognizes a unique sequence of 4-6bases along the DNA strand and cuts the strand at these sites - the first step in aprocess called restriction mapping. These smaller, specific sections of anorganism’s DNA can then be studied in detail and an outline of the wholegenome can be constructed. This procedure is one of the most important inmodern biology.The small fragments of DNA are separated by gel electrophoresis. Themovement of the fragments will always be towards the positive electrodebecause DNA is a negatively charged molecule. The fragments move throughthe gel at a rate that is determined by their size and shape, with the smallestmoving the fastest.DNA cannot be seen as it moves through the gel. A loading dye must be addedto each of the samples before it is pipetted into the wells. The progress of thedye can be seen in the gel. It will initially appear as a blue band, eventuallyresolving into two bands of different colors.The faster moving, purplish band is bromophenol blue dye that migrates at

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Jul 23, 2017
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