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Activity 2 - Gel Electrophoresis of Dyes

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  Share  Activity 2: Gel Electrophoresis of Dyes Introduction This experiment will teach students how to prepare and load an electrophoresisgel. They will then run the gels in an electrophoresis system to separate severaldyes that are of different molecular sizes and carry different charges.This technique is fundamental to many of the procedures used in biotechnology. Objectives 1. Understand how gel electrophoresis is able to separate molecules.2. Learn how to use a micropipet to load a gel.3. Review safety considerations when working with an electric current.4. Determine the components of an unknown dye mixture. Materials Samples of the following dyes:0.25% Bromophenol blue0.25% Janus green1.00% Orange G0.25% Safranin O0.25% Xylene cyanol0.25% Unknown mixtureMicrotube rackElectrophoresis gel box and power supply1 gel tray with 6-8 tooth comb250-ml beaker or graduated cylinder 20-μl micropipette with tipsGlovesPaper to cover lab bench60°C water bath or microwave oven0.7% agarose solution (enough for 35 ml per gel)Container with TBE buffer (1X)Plastic bag for disposal of wasteDistilled water   Advance Preparation Make the 0.7% agarose gel solution as follows: To make 100 ml of gel, which is sufficient for 3 gels, weigh out 0.7 g of agaroseand place into a 200- to 250-ml glass beaker or flask. Add 100 ml of 1X TBE(Tris-Borate-EDTA) buffer. Heat in the microwave for 30 seconds at a time,shaking gently each time, until the agarose is completely melted. Alternatively,the solution can be heated on a hot plate, with occasional gentle shaking, untilthe agarose is melted. Keep warm if the class will use it within a half hour.Otherwise, allow the solution to cool and solidify. Cover and keep in therefrigerator. Lessons andLabs Plant Biotech Activities HomeWhat is DNA?DiseasesHistoryHealthEnvironment ActivitiesConclusions  APS > Education > K-12 > Lessons and Laboratories > Classroom Activities in PlantBiotechnology > Activity 2 - Gel Electrophoresis of Dyes  Day of class: Loosen the lid from the container of 0.7% agarose. Immerse the bottle in adistilled hot water bath to melt the liquid. Alternatively, melt the agarose in amicrowave and then keep warm in a hot water bath. Do this step well ahead of time and keep the agarose warm until needed by the students. Students willneed to handle the bottles so have potholders available. After the lab, the TBE buffer solution may be poured back into the container andreused for the Restriction Digest Laboratory. Information for the teacher  Using a Micropipet  A micropipet is a very delicate, expensive instrument that is used to dispense anextremely precise and very small volume. It is important to know the volumelimits of the micropipet that you are using and to never dial either lower or higher than these limits. The volume settings on the micropipet are generally read fromthe top to the bottom of the number dials. Often the numbers before the decimalplace will be in a different color to those that come after it. For example, on a200-µl micropipet a volume set at 63.5 µl might have the 6 and the 3 in blacklettering but the 5 might be in red lettering.The micropipet is designed to be held in one hand. Set the micropipet to thedesired volume by turning the volume adjustment knob. Using adisposable tip that is the correct size for the micropipet push the shaftof the micropipet firmly down into the sleeve of the tip until a firm sealis made.To aspirate (pull in) the liquid, first depress the plunger to the firststop, place vertically into the liquid to a depth of about 2-3 mm andslowly release the plunger until it returns to its up position. Now youcan move the micropipet out of the liquid and over to the agarose gelwell or other vessel.Place the tip against the wall of the well or other surface and begin toslowly depress the plunger, but this time you must move the plunger all the way to the second stop in order to expel all the liquid.While still holding the plunger in the depressed position, move it out of the well or other vessel and then allow the plunger to return to its normal position. If theplunger is released while it is still in the well it will aspirate the liquid that you justdeposited there!Discard the tip into an appropriate waste container by pressing the ejector buttonwhich is found near the top of the micropipet. Always use a fresh tip for eachsample to avoid cross contamination of the samples. Buffer Solution: Tris/Borate/EDTA (TBE) buffer is commonly used in electrophoresis systems.This salt solution conducts the electric current and controls the pH of the solutionduring separation of DNA fragments, or in this case, dye molecules. Dilute thestock solution as necessary with distilled water to make a 1X solution. Distilled Water:  Minerals in regular tap water will quickly stain equipment. Please rinse and air dry both the gel trays and gel boxes in distilled water. Be careful not to dislodgethe wiring at the base of the gel box during this process. Gel Disposal: When lab is complete, collect all gels in the plastic bags and dispose of in trash. Use of Power Supplies The power supply produces a voltage that is high enough to cause severeelectrical shock if handled improperly.Do NOT plug power supply into wall receptacle until the safety cover ispositioned on the cell and all other electrical connections are properlymade. This unit uses a 3-wire grounded plug. For safety reasons, it should NOTbe used with 2-wire receptacle with a conversion plug. Do not operate in a damp or humid environment; any condensedmoisture may short out electrical components. Make sure that allelectrical equipment is dry. Inspect all power cords, patch cords, banana jacks and plugs for anydefects, such as cracked and dried-out insulation and loose or wobblybanana jacks or plugs. Do not come in personal contact with or allow metal or any conductivematerial to come in contact with reservoir buffer or the electrophoreticcell while power supply is on. The power supply may continue to produce some voltage even when thepower has been turned off. To eliminate any risk associated with thisevent, follow all required steps given in the procedure. Whendisconnecting the gel box, be sure that the leads do not touch eachother, come in contact with the buffer solution, or otherwise create anyhazardous electrical condition. Notes  Agarose is a substance derived from seaweed that forms a jelly-like matrix whendissolved in liquid. During electrophoresis an electric current is created throughthe agarose and molecular fragments can move through the agarose betweenthe two electrodes.The size of the pores in the gel and the size of the fragment trying to move willdetermine the rate at which each fragment progresses. The direction in which afragment moves is determined by the charge that the fragment carries.When using this protocol the granular dyes should be made up in the followingconcentrations:For all except Orange G use 25 mg of dye, add either 10 ml of 60% glycerolsolution or 4 g of sugar and make up to 10 ml with distilled water.For Orange G use 100 mg of dye and make up as above. The Orange G is only50 bp in size and is easily run off the end of the gel. It runs far ahead of theBromophenol blue which is the easiest dye to see.For the liquid dyes, if necessary, dilute with the 60% glycerol solution to therequired dilution.  Student Activity - Gel Electrophoresis of Dyes In this experiment you will be using electrophoresis to separate dye sampleswhich have different sizes and charges.  Precautions STUDENTS: Check with your teacher to be sure that you understand all of thesafety instructions for using this equipment. Objectives 1. Understand how gel electrophoresis is able to separate molecules.2. Learn how to use a micropipette to load a gel.3. Review safety considerations when working with an electric current.4. Determine the components of an unknown dye mixture. Procedure 1. Put on gloves. You may be sharing dye samples and the agarose gel withanother lab group. 2. Seal each end of the gel tray with laboratory tape. Place the plastic combinto the middle of the tray. Go to the hot water bath and get the bottle of melted agarose. 3. Carefully pour the agarose into the gel tray until it is approximately 1/3 of the way up the teeth of the comb. This should use about 30 ml of agarosesolution. Make sure that there are no bubbles in the gel. 4. Let the gel harden without disturbing it for about 10 minutes.5. Carefully remove the comb from the gel by pulling straight out of thesolidified gel. Remove the tape from the ends of the gel tray. 6. Place gel into electrophoresis unit. Add 150 ml 1X TBE buffer to completelyfill the box and to cover the top gel surface with about 2 mm of buffer.Note: At this point the gel box can be covered and left until the nextday if necessary.7. On the gel load 5-10 µl of each dye into a well. Keep track of which dyegoes into which well on a notebook sheet. Use a new tip for each dye andbe careful not to puncture the bottom of the well. Dyes to be used:Bromophenol blue

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Jul 23, 2017
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