ELECTROPHORESIS OF SPOOLED DNA 1 An Introduction to Agarose (Horizontal) Electrophoresis

Transcription

1 ELECTROPHORESIS OF SPOOLED DNA 1 An Introduction to Agarose (Horizontal) Electrophoresis INTRODUCTION This laboratory will demonstrate the basics of horizontal electrophoresis and the theory behind the separation of DNA molecules in an agarose gel. Agarose gel electrophoresis is most commonly used to separate DNA fragments, but may also be used to separate proteins. How a molecule migrates through a gel is dependent upon its size, electrical charge and shape. Smaller molecules will travel faster through pores of an agarose gel, whereas larger molecules will migrate more slowly. Negatively charged molecules will run toward the positive (red) electrode of the electrophoresis chamber, while positively charged molecules will migrate toward the negative (black) electrode. Migration of a molecule may also be affected by the concentration of agarose in the gel and the type of buffer used. These properties of electrophoresis can be demonstrated using spooled chromosomal DNA and DNA size standards. Students should be able to form basic hypotheses about the size and charge of the chromosomal DNA molecules when compared with the DNA size standards. PURPOSE The aim of this lab is to familiarize the students with the basic elements of agarose electrophoresis. The students will be able to: a. pour an agarose gel. b. load DNA samples into a well and perform electrophoresis. d. develop a hypothesis about the migration of DNA in an agarose gel. c. analyze the results of DNA isolation or other types of DNA manipulation. MATERIALS Day 1 Day 2 - agarose powder - electrophoresis chamber & power supply - 1x TBE buffer - DNA samples - hot pad or mitt - DNA loading dye - clean beaker or flask - DNA size standards for electrophoresis - microwave (or hot plate & stir bar) - micropipettor and tips - gel casting tray and gel dams or masking - paper towels tape Day 3 - methylene blue stain & large weigh boats or staining dishes - light box or overhead projector - plastic wrap 1 Adapted from Electrophoresis of Spooled DNA: An Introduction to DNA Electrophoresis (1996) (8 May 2002) Westminster College SIM 1

2 SAFETY PRECAUTIONS Day 1: To dissolve the agarose properly, the agarose and buffer solution must come to a boil. It is important to have a hot mitt to handle the flask containing the agarose solution. If you are using a microwave to heat the agarose solution, be aware of superboiling. It may look as though the solution is not boiling, but when you touch the flask, the liquid gushes up and out of the flask. To avoid this, let the solution microwave for sec, then take the flask out with a hot mitt and swirl the solution (keeping it away from your face or body). Finally, if the solution has not cooled enough, there is a possibility of steam burns when actually pouring the solution into the casting tray. While some protocols advise cooling the melted agarose to 55ºC before pouring it into the casting tray, this often leads to premature solidification and formation of clumps in the gel. One solution to this problem would be to cool the gel in a 60ºC water bath for 5 minutes. An easier solution is to simply let the flask cool at room temperature for 4-5 minutes, swirl it to re-mix the solution, and then pour it into the casting tray. Use of a hot mitt is still strongly advised. Day 2: Exercise caution when using the power supply. The area around the power supply and the electrophoresis chamber should be dry. Be sure the lead is sitting on the electrophoresis chamber properly and all the connections are in place before turning on the power. Likewise, the power supply should be shut off before disconnecting any of the electrical leads. PROCEDURES Day 1- Gel & Sample Preparation 1. Prepare the gel casting tray by taping the open ends of the tray firmly with masking tape. Use your nails to press down on the edges making sure that the ends are sealed and no leaking will occur. 2. Determine the volume of 1X running buffer required for the casting tray. For example, a 7 X 7 cm Bio-Rad casting tray requires 40 ml of 1X buffer. Measure out the required amount of buffer using a graduated cylinder and then pour it into a 125 ml or 250 ml Erlenmeyer flask. (If making up a large batch of agarose for casting numerous gels all at once, multiply the required volume of buffer for one gel by the number of gels being made plus two extra gels.) 3. Use a scale and small weigh boat to measure the amount of agarose required for a 0.8% agarose gel. For example, 0.32 g of agarose needs to be measured if using 40 ml of buffer and a 7 X 7 cm casting tray. (If making up a large batch of agarose for casting numerous gels all at once, multiply the required amount of agarose for one gel by the number of gels being made plus two extra gels.) 4. Heat the agarose solution in a microwave for one minute on high. The solution should just begin to boil. (If using a hot plate, heat with intermittent stirring until the solution begins to boil.) Carefully remove flask and gently swirl at arms length. Reheat the agarose for another seconds in the microwave and then swirl again. Repeat this Westminster College SIM 2

3 process (20-25 seconds) as needed until agarose grains are completely melted (2-3 more times should suffice). Then let cool at room temperature for 4-5 minutes. 5. Pour the hot-warm liquid agarose into the casting tray. Immediately place the gel comb into the end slot. How the comb is aligned is dependent on the apparatus being used. The most important factor is that the comb does not touch the bottom of the casting tray or the wells will not hold the sample. 6. Allow the gel to cool undisturbed for about 15 minutes. The agarose solution will become cloudy and firm to the touch when it is completely hardened. Placing the casting tray on a cool surface will decrease the gelling time. 7. The gel, still in the casting tray, can be stored for several days in a refrigerator if it is wrapped in plastic wrap or placed in a sealable bag. The combs may be removed prior to storage. Remove remove the comb slowly and carefully so that the bottom of the wells do not rip. 8. To prepare the DNA samples, DNA loading dye needs to be added to a small aliquot of the spooled DNA resuspended in buffer. The DNA concentration is unknown, so aliquots of different amounts are recommended. For example, from one stock of spooled DNA prepare three tubes containing 2.5 µl, 12.5 µl or 25 µl of the sample. Bring each tube to a total volume of 25 µl (for example, add 22.5 µl distilled water to the tube with 2.5 µl DNA solution). For each 10 µl of DNA sample, you will want to add 2 µl of DNA loading dye to be sure the samples will load into the wells easily. So 5 ul of loading dye must be added and thoroughly mixed with each 25 ul sample of spooled DNA. Day 2 Electrophoresis 1. Carefully remove the tape from the ends of the casting tray and place the gel into the electrophoresis chamber. Orient the gel so that the wells are closer to the negative (black) terminal. 2. Fill the electrophoresis chamber with 1X TBE. Be sure that there is just enough buffer to completely submerge the agarose gel. 3. The teacher will demonstrate the proper way to hold a micropipette, fill it with sample and dispense the sample into a well. 4. Fill the pipet with the entire 25 ul of DNA sample. Place the tip over the top of one of the wells. The tip should be submerged in the buffer at this point. Holding the pipet steady, gently dispense the sample into the well. The loading dye in the sample will allow the sample to sink into the well. Do not place the pipet tip directly into the well or you will risk poking a hole in the side or bottom of the well, and your sample may leak out of the gel. A new pipet tip should be used for each sample. If possible, each student should have a turn loading a well. Remember to load the DNA size standards. Westminster College SIM 3

4 5. Record the order in which the samples are loaded, either left to right, or top to bottom. 6. Make sure that the area around the electrophoresis chamber and power supply is dry. The teacher will connect the electrical leads (black to black and red to red) and turn on the power supply. A voltage of 100 V is optimal. More than 150 V is not recommended, as the agarose gel can melt. Check the gel after approximately 5 min. to make sure the sample is migrating through the gel and in the correct direction. Allow the gel to run for a total of minutes. The dye in the loading solution can be used to gauge the progress of the sample in the gel. The gel may be stained immediately or stored in plastic wrap and refrigerated until the next class period. Day 3 - Gel Staining & Analysis 1. Place the gel into an old plastic container and cover the gel with a solution of 0.025% methylene blue for min. This solution will stain both the gel and the DNA. Destain the gel with several changes of warm (37ºC) distilled water, until the DNA becomes visible against the clear background of the gel. 2. Place the gel on a piece of plastic wrap. View the gel on a white background with the light shining from beneath. Either a light box or overhead projector can be used for this purpose. If these are not available, a bright light over the gel will work as well. Westminster College SIM 4

5 ELECTROPHORESIS OF SPOOLED DNA Analysis of Results Name: 1. Draw a picture of the gel using graph paper. Try to reproduce the position of the DNA size standards and the DNA smear as closely as possible. 2. Utilizing the DNA size standards, determine the size range of the DNA molecules. What is the smallest DNA molecule that you can detect? What is the largest? Are most of the DNA molecules fairly intact or is their degredation of your spooled DNA sample? 3. What is the charge of the DNA molecules? How does electrophoresis utilize this charge to help the DNA migrate through the agarose gel? 4. Large DNA molecules have a stronger negative charge than smaller DNA pieces. Using this information and your gel, which is more important in determining how far the DNA will migrate the size of the DNA molecule or its charge? Westminster College SIM 5

6 TEACHER NOTES 1. The actual experimentation portion of this lab is written for three 50 min class periods, but could be modified for two class periods. This would require that the teacher prepare the gel, DNA samples, DNA size standards and the electrophoresis chamber prior to class. The gel would need to be loaded and electrophoresis (at V) started as soon as class has begun. While the gel is running, the principles of electrophoresis could be taught. After about min, electrophoresis could be stopped and the gel stained with methylene blue. The destain procedure should be completed after class and the gel wrapped and stored in a refrigerator until the next class period. 2. If running more than one gel in a class, there are variations which would allow the students to compare the effect of external variables on the separation of the DNA molecules. a. Pour gels of different densities. Select 3 different percentages of gel and run all three for the same amount of time at the same voltage. (0.8% agarose, 1.0% agarose, and 1.2% agarose are densities commonly used in research labs.) Then have the students compare the differences in the migration of the DNA size standards for each gel. Which percentage has the fastest rate of migration? b. Use different electrophoresis buffers. In addition to TBE buffer, TAE (Trisacetate, EDTA) and TPE (Tris-phosphate, EDTA) are also commonly used. By running 1% agarose gels at the same voltage, it is possible to compare the effects of the buffers on DNA separation. If TPE buffer is used, a lower voltage (60-70 V) will need to be used, as a higher current in this buffer tends to melt agarose gels more easily. Westminster College SIM 6