Slide 1Setting Up an Electrophoresis GelElectrophoresis involves:Determining the size of gel neededPreparing Your Gel TrayMaking the Gel SolutionSlide 7Slide 8Slide 9Pouring the GelRemoving Bubbles that FormPlacing the Gel Tray into the Tray BoxSlide 13Preparing the DNA SamplesLoading Your Gel (Part 1)Loading Your Gel (Part 2)Attaching Your Tray Box to a Power Supply: Part 1Attaching your Tank to a Power Supply: Part 2Analyzing Your GelWhere to Get More Information01/14/19 1ElectrophoresisThe purpose of electrophoresis is to separate molecules of DNA, RNA or protein.Separation can be based upon:1) Size2) Shape3) Isoelectric point (protein only).Separation is achieved by using a matrix (gel) and an electromotiveforce to propel the molecules through the gel .01/14/19 2Setting Up an Electrophoresis Gel•To run a gel electrophoresis you will need•Gel tray•Gel comb•Electrophoretic gel box•Agarose powder•Buffer solution•Dye •Electric field01/14/19 31. Preparation of the gel tray2. Melting the agarose in a buffer solution3. Addition of dye to the molten agarose solution (optional)4. Pouring the molten agarose into the casting tray with a comb5. Letting the gel solidify6. Submerging the solid gel in buffer solution in the gel box7. Removing the comb to reveal the wells in the gel8. Loading the samples into the wells9. Applying the electric current and “running” the gel 10. Analyzing the samples separated in the gel Electrophoresis involves:01/14/19 4Determining the size of gel needed•One must first decide what size gel will be run. In general:–For a small gel tray, about 50 ml of gel is needed–For a medium gel tray, about 100 ml of gel is needed–For a large gel tray, about 200 ml of gel is needed(note: these are estimates, the volumes will vary with the apparatus used!smallmediumlarge01/14/19 5Preparing Your Gel Tray•To form a mold for the hot liquid gel, the tray must be taped on the open sides•To accomplish this pull one piece of masking tape over the open side leaving about 1 cm on the bottom to fold it over thus making a good seal•Pour the gel in and insert the gel comb•Let the gel cool •Remove the tape and the comb and the gel is ready for useNote: some electrophoresis apparatuses come with gel-casting boxes01/14/19 6Making the Gel Solution•Weigh out the appropriate amount of agarose powder–For example 100ml of a 1.6% Agarose solution will need 1.6 grams of agarose •Pour the agarose powder into a Erlenmeyer flask (the flask should be at least 2X the volume needed to avoid boiling over when the solution is heated) –For example 100 ml of gel solution will be made in a 250 ml Erlenmeyer flask01/14/19 7Agarose PowderThis is commercially available Has the consistency of fine sugarNon-toxic Weighing out the powderUse a weigh boat and scaleTare the scale with the empty containerAdd an appropriate amount of agaroseMaking the Gel Solution01/14/19 8Add the appropriate volume of buffer to the agarose powder• Commonly TAE or TBE are used for buffers.• The buffer controls the pH of the solution and the salts in the buffer improve conduction of electricityTAE and TBE buffers are available commerciallyMaking the Gel Solution01/14/19 9•Cover to prevent water loss and microwave initially at high power for 60 seconds per 100 ml of solutionRemove and carefully stir by swishing the solution (the solution may be superheated so wear oven gloves!)Return solution to the microwave and keep heating and swishing for 30 second intervals until all the agarose powder has dissolvedMaking the Gel SolutionNote: this can also be done using a hot-plate!01/14/19 10Pouring the Gel•After the gel has cooled (to 60C) and before it starts setting, the gel solution must be poured into the prepared gel tray •Pour slowly to ensure no bubbles form•Pour until the comb teeth are at least 50% covered by the gel01/14/19 11Removing Bubbles that Form•If a few bubbles form, use a disposable pipette tip to move the bubbles to one side of the gel tray – keep the bubbles away from the middle of the gel and the comb.•Bubbles can interfere with DNA progressing evenly through the gelLet the gel set until it become opaque01/14/19 12Placing the Gel Tray into the Tray Box•Carefully remove the tape from your gel tray•Place the gel tray (with gel) in the tray box •Add the Tris buffer (same buffer that was used to form the gel) into the tray box until the level of buffer is 4 or 5 mm above the gel•Remove the comb carefully- the holes made by the comb will become the wells. This is where the DNA samples are placed01/14/19 13Remove the tape carefully from each end of the gelCarefully remove the comb.01/14/19 14Preparing the DNA Samples•To properly run a gel, one must add a loading buffer to the samples before they are placed into the wells. The purpose of the loading buffer is three-fold •To track the progress of the electrophoresis •To make it easier to load the wells•Add to the density of the DNA sample so the solution will sit in the well•Decide on the sample order (and take note of this in lab notebook!)•Put up to 20 µl of a DNA sample in a clean tube•Add 5 µl of loading buffer to the sample01/14/19 15Loading Your Gel (Part 1)•Place a piece of black paper under the tray box to help visualizing the wells.•Carefully withdraw the sample from the tube with a pipettor.•Gently pipette the sample into the well•Keep track of the order in a notebook!01/14/19 16Loading Your Gel (Part 2)•Remember to leave some wells free for the ladders•Ladders are size standards that assist in identifying the size of a sample after the gel has run•You should leave a space between your samples and the ladders•The last wells on either side of the gel are usually left empty as these lanes do not run properly01/14/19 17Attaching Your Tray Box to a Power Supply:Part 1Place the cover on the tray box and attach the electrodes Remember “DNA runs to Red”The electrode near the wells attaches to the negative terminal (cathode) and the other electrode attaches to the positive terminal (anode).Once the wires are attached, turn on the voltage. As a general rule: 5 Volts per cm (distance between electrodes on the tank)Distance between electrodes01/14/19 18Attaching your Tank to a Power Supply:Part 2•In general up to 100 Volts is used for a small gel and up to 200 Volts for a medium gel•If current is running from the cathode to