pglo MUTAGENESIS SESSION FOUR-2

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1 pglo MUTAGENESIS SESSION FOUR-2 Use of Agarose Gel Electrophoresis In Selecting The Best Mutants For Further Study In general, electrophoresis is a method used to separate cell fractions, proteins, nucleic acids or chromosomes. There are many different protocols used in electrophoresis but all take advantage of an inert support matrix (e.g., starch, agarose or polyacrylamide) and an electric field. Molecules move in this electric field. In agarose gel electrophoresis, agarose serves as a molecular sieve to separate the molecules according to their size, shape and net ionic charge. We will be making extensive use of the agarose gel electrophoresis method in our work here for the separation of DNA fragments. DNA is negatively charged at neutral ph and thus moves towards the anode (positive pole). To elucidate the use of this technique, suppose we digest a long piece of a DNA with a restriction enzyme (RE). How do know how many fragments are produced and what their sizes are? Fragments can be separated on the basis of their size by the technique of agarose gel electrophoresis. The sample is placed at one end of an agarose gel and subjected to electrical current. Small fragments move very fast in the agarose, while large fragments move at a slower rate. In this way fragments are separated into distinct bands, the sizes of which can easily be estimated if a sample of fragments of know sizes is run alongside the unknown sample. Agarose is a polysaccharide powder that when mixed with water, boiled and then cooled, will turn into a gel-like substance. This gel is a complex network of fibrils through which DNA fragments must pass. The distance between the fibrils or the pore size is determined by the concentration of agarose used (i.e., a 20% agarose would hardly let even very small DNA fragments to pass through while a 0.2% agarose is rather flimsy and can let very large DNA fragment to travel through at a fast speed). Therefore, controlling the concentration of agarose based on the sizes of the DNA molecules is important. The shape of the molecules also plays an important role in its relative migration distance in an electric field. Compact molecules naturally move more rapidly than loose molecules. Two other important factors are the amperage of the electrical field and the ionic strength of the electrophoresis buffer. With higher amperage and/or buffers of high ionic strength, the molecules move faster in the field. However, too high a voltage, amperage, or ionic strength of the buffer would cause heating of agarose and loss of its natural matrix, and even denaturation of DNA. Electrophoresis is conducted in a Plexiglas apparatus composed of a tray in the middle section where the agarose gel is poured and two buffer compartments or tanks at either side which are connected by platinum wires to the current. Many sizes or shapes of the apparatus are now commercially available or can be constructed in the lab. One should make sure that, when a gel is running, the apparatus is well shielded and there are no exposed parts that could impart an electric shock. 1

2 The agarose gel is made by adding the powdered agarose to an electrophoresis buffer, boiling it to melt the agarose and pouring it into trays that already have one or two combs placed in the proper spots. The combs make indentations or wells in the agarose when it solidifies and provide depressions into which samples can be loaded. We usually add a dye (called loading dye) to the samples before transferring our samples to the wells. The loading dye has two main functions. One is that it makes us able to visually follow the front and the other is that it contains a high concentration of glycerol that pulls the samples to the bottom of the wells in the agarose. If the loading dye is not added, we would be unable to know when to turn the current off and whether or not the fragments are moving in the agarose medium at all. Also, mixing of the samples may occur because they may not stay in their own wells. Note that the loading dye is different from the DNA dye that we should use to stain the DNA fragments in the gel (see below). Staining of DNA in an Agarose Gel There are two ways to stain an agarose gel: by adding the DNA stain directly into the gel, or by running the gel without any DNA stain and then immersing the gel into a solution of DNA stain. Two such stains are ethidium bromide (EtBr) and SYBR Gold. DNA stains are actually intercalating agents that lodge themselves in between DNA bases and since they are fluorescent under UV irradiation (when the gel is exposed to UV), the DNA fragments will be visually observable as colored bands in the gel and we can photograph the gel to obtain a permanent record. By comparing each band with a standard, run in parallel in the gel, we can approximate the size of each fragment. Unfortunately, EtBr is a highly mutagenic/carcinogenic agent and extreme care should be practiced in its handling. Its waste disposal is another problem that many molecular laboratories have to deal with. Recently new dyes have been produced by chemical companies that are as good as or better than EtBr in resolving electrophoretic bands with much less toxicity. Since student and staff safety is one of our priorities in this lab, we have replaced EtBr with a dye called SYBR Gold and we have obtained excellent results with this dye. We also tried a newer and safer product, recently introduced to the market, called SYBR Safe but unfortunately the results were not as good as SYBR Gold. WARNING: Although the Ames test has shown very low toxicity for SYBR Gold and SYBR Safe, we still caution you to wear gloves and pay special attention to safety methods and regulations since any intercalating agent can be, by nature, mutagenic and quite hazardous. SYBR Gold is highly concentrated. It should be diluted 10,000 times before use. We use 4 µl of concentrated SYBR Gold in our 70 ml gel. If the gel is run without adding SYBR Gold, 10 µl of SYBR Gold should be added to 100 ml of water and the gel should be immersed in this solution for about 30 minutes, after which time, the gel can be photographed. No de-staining with water is required and the stain can be covered with aluminum foil and placed in the refrigerator and reused 2-3 times. 2

3 Laboratory Supplies Standard DNA, 1 Kb ladder (100 ng/µl) pglo, 50 ng/µl Eppendorf tubes Microfuge Loading dye Bio-Rad gel apparatus + 8-tooth comb Sterile distilled water Power supply Agarose Balance Heat-resistant gloves Electrophoresis buffer, TAE (1X) Flask, 125 ml Spatula Graduated cylinder, 100 ml Graduated cylinder, 1 L Microwave oven SYBR Gold Tape, roll Photography equipment Pipetmans, one set of P20 and P200 Pipet tips, large and small Pipetman holder 1 beaker/table 2/lab 1/table 1 tube/table 1/table 1 bottle/table 1 pair/lab 5 L/lab 1/group 2/lab 2/lab 1/table 1/group 1 box of each/group 1/group Procedures 1. Get ice and label 4 new Eppendorf tubes A-1 and A-2 and G-1 and G-2 and place them on ice. Transfer 2.5 µl of plasmid DNA into respective tubes and store the original tubes at 20 C till the next session. 2. Add 7.5 µl of sterile water to each tube. Also prepare a tube of 1 Kb ladder by adding 2.5 µl of the standard and 7.5 µl of sterile water. Place this tube on ice, too. 3. Add 2.5 µl of 100 ng/µl pglo and 7.5 µl of sterile water to an Eppendorf and mix. Label the tube pglo and place on ice. 4. Add 2 µl of loading dye to all tubes, mix and spin for a few seconds to bring all liquid to the bottom of tubes. Leave tubes on ice until needed. We add the loading dye (bromephenol blue) to our samples to make it easier to visually follow the fronts. 5. Obtain a complete Bio-Rad gel apparatus which consists of a gel base and a gel caster. See demo by your TA to set up the gel caster and the UV-transparent gel tray. Make sure the cam lever is tightened. Place an 8-tooth comb at one end into its slot. Make sure the gel caster is on a level surface. 6. Weigh 0.56 g of agarose in a weighing boat and pour into a 125 ml flask. Add 35 ml of 1X TAE buffer to the flask in a way to wash all the agarose down to the bottom of the flask. Swirl the flask and make sure all the agarose is suspended and there are no undissolved lumps. 3

4 7. Place the flask inside a microwave and choose a 2 minutes heating cycle. However, less than one minute is enough to bring the TAE to a boil. Start the microwave oven and watch the liquid inside the flask carefully for signs of boiling. Wear special heatresistant gloves. After a few seconds of boiling, open the door and take the flask out and swirl a few times to make sure all the agarose is dissolved. Note: The flask will be very hot, so pick it up with special gloves provided next to the microwave. 8. Place the flask back into the microwave and start the oven again. Again watch for any sign of boiling. When you see it, let it go a few more seconds and then stop the microwave. Wear heat-resistant gloves, take the flask out and swirl gently to mix. Try not to introduce bubbles in the solution. Set the flask down on the bench top. Note: The flask will be very hot, so pick it up with special gloves provided next to the microwave. 9. When there are no bubbles at the surface, add 35 ml more of TAE to the flask and swirl the flask very gently to mix. Again be careful not to introduce bubbles to the mixture. 10. Your TA will wear gloves and using a P-20 adds 4 µl SYBR Gold to the middle of agarose. Then swirl the flask very gently 10 times one way and 10 times the other way 2-3 times to completely mix the contents. [TA: Please make sure you discard the tip into the biohazard bin.] 11. Pour all the contents of the flask into the gel tray and let the agarose solidify. You should not move the gel caster until the agarose hardens (about 10 minutes). Meanwhile, use hot tap water to rinse out the flask and place it on a paper towel to drain and dry. 12. Fill the electrophoresis chambers of the gel base with TAE until the two chambers become connected. 13. Wearing gloves, loosen the cam lever on the gel caster and transfer the gel tray with the hardened agarose and the comb into the gel base. When it is properly placed, it sits snugly into its slots. Do not remove the comb yet. 14. Add TAE so it will cover about 1-2 mm over the gel. Now, gently remove the comb and wash under tap water and let dry. Your gel is now ready to be loaded with samples. 15. Turn the gel so the wells would be on the top. Use a P-20 to load one group s samples into the top wells, starting with the standard being loaded in the far left side. Make sure you keep track of sample sequences on the gel and make notes in your notebook (e.g. Std, pglo, A-1, A-2, G-1, G-2). 16. After all the samples have been loaded into the gel, place the lid on the chamber making sure the negative pole is the one connected close to the wells. The negative wire is usually black. 17. Set the Voltage to 100 volts, (the maximum milliamperes to 400, if the power supply is equipped for it) and the Time to 40 minutes and press run on the power supply. You should see bubbles emanating from the platinum wires inside the chamber. 4

5 Note: Some power supplies do not have a setting for the maximum tolerable milliamperes. 18. Check the leading dye movement to make sure that everything is going fine. If you do not see any dye color appearing within 5-10 minutes, check to make sure your connections are secure and correctly connected. 19. When the run is complete, turn off the power supply, gently remove the lid and take out the gel tray. Note: The lid is very fragile and breaks in two very easily. Make sure you handle it with care. 20. Place the gel tray on a UV transilluminator and photograph the gel. Your TA will give a demo of how to best do this and print a copy of the gel. 21. Look at the gel photograph and see if everything is according to your expectations. Based on the bands and amounts of DNA, choose one best A and one best G mutant for your group to be used in the next session. Use of any section of this Lab Manual without the written consent of, Dept. of Biology, University of Pennsylvania is strictly prohibited. 5

6 Results of pglo Mutagenesis Lab Exercise pm: Session Four Name Date Section Name of partner Paste your gel photograph below and explain the bands that you observed. Notice the sizes of your plasmids. The 1 kb standard ladder is given below. Which mutants are you choosing for further study? Justify your answer kb