How to load and run an Agarose gel PSR Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from100 bp to 25 kb. This protocol divided into three stages: 1. Preparing gel with an agarose concentration appropriate for the size of DNA fragments to be separated; 2. loading the DNA samples into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation; 3. visualizing DNA fragments with SYBER green incorporated into the gel and electrophoresis bufferd with UV light. Golchin. A 4/5/2016
Workflow Preparation of the Gel Loading Samples and Running Observing Separated DNA fragments Detailed procedure Materials product Cat. No. or LOT.No Gel box/tank, gel tray, gel comb(s), power supply, UV viewing table, camera (or more sophisticated gel viewing and image - producing equipment), pipettes, pipette tips, gloves, goggles, 250 ml Erlenmeyer flask, DNA ladder/marker, 1 kb #SM0311 Agarose #R0801 SYBR Green I LOT: 1567474 Gel Loading Dye #R0611 TAE Buffer (40 mm Tris-acetate, 1 mm EDTA) DNA samples you want to run - 60
Don t forget to leave at least one well for DNA ladder/marker There is no difference between a double row gel and a single row gel except that in a doublerow gel you just have to be sure that your top samples don t run down into the bottom section. For best results, use the same concentration and batch of buffer in both the gel and the gel box. Method Preparation of the Gel 1. Figure out the best concentration of agarose to use based on what size DNA you want to visualize. The DNA will show up pretty much on any gel, but the resolution will be better if you follow the table below. On that note, however, don t worry about measuring exactly when making the gel, because a small difference in concentration doesn t matter that much. Only worry about exact measurements when you need to be super consistent between gels (e.g. RFLP). Also, agarose is a little expensive, so don t make a 2 or 3 % gel unless you really need to separate fragments that only differ in size by a few base pairs. Gels less than 1% are flimsy, however, so handle them carefully. The more concentrated a gel is, the stiffer and easier to handle it is. Good rules of thumb are: to look at PCR products make a 1.5 % gel and to look at genomic DNA make a 1% gel. Separation of DNA in agarose Efficient range of Agarose in gel (percent) separation of linear DNA molecules (kilo-bases) 0.3 5-60 0.6 1-20 0.7 0.8-10 0.9 0.5-7 1.2 0.4-6 1.5 0.2-4 2.0 0.1-3 2. Prepare an adequate volume of electrophoresis buffer (TAE 1X) to fill the electrophoresis tank and prepare the gel. 3. Make the melted agarose solution. The small gel is made with 35-40 ml of buffer and the large gel is made with 80-100 ml of buffer. The percent agarose is calculated in g/ml as in the following: To make a 1% gel in the small gel tray, use 0.35 grams of agarose in 35 ml of buffer. 0.35g / 35ml = 1% Weigh out the appropriate amount of agarose using the designated agarose weigh boat that is on the first shelf above the nano drop bench, at nano-bio medicine lab. 61
4. Add running buffer to the agarose containing flask that is on the second shelf above the microwave bench, at molecular-medicine lab. Swirl to mix. The most common gel running buffers are TAE (40 mm Trisacetate, 1 mm EDTA) and TBE 1X (45 mm Trisborate, 1 mm EDTA).( RCPN uses TAE1X) 5. Microwave for several min until the agarose is completely dissolved and there is a nice rolling boil. If using a microwave, heat the solution for several short intervals - do not let the solution boil for long periods as it may boil out of the flask). Use a paper towel or flask holder to grab the flask. Note: Caution HOT! Be careful stirring, eruptive boiling can occur. CAUTION: Wear gloves, because SYBR Green binds to nucleic acid, which your cells have a lot of! 62
6. While still fluid, add 1 µl of the SYBER Green I per each 10 ml of gel solution. Mix thoroughly.( Pre-staining with SYBER Green I) 7. Pour the agarose into the gel tray, with combs in place. Remove any air bubbles. 8. Gel should be ready in about 10-15 min (cloudy and firm). Loading Samples and Running 1. Add loading dye to the DNA samples to be separated. Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel. Remove any air bubbles. 63
2. Mix by pipette 5 μl of sample with one loading dye dot and add to the well in the gel; change tip between samples. Load 5 μl of ladder and loading dye in one well (center well is recommended). 64
3. Fill gel box with TAE 1x until the gel is covered. (about 250 ml) 4. Don t forget to load a DNA ladder into at least one of your wells. DNA ladders, also called molecular weight markers, are used as a standard so that you know how far DNA of a known size migrates in your gel. We have 3 different ladders: a) 100 bp ladder good for fragments that are 100 bp 1 or 2 kilobases (kb) b) 1 kb ladder good for fragments larger than 500 bp c) Quant-it Ladder quantitative ladder for determining approximate DNA concentration. 5. Close the lid of gel tank. 65
6. Attach the leads of the gel box to the power supply. Turn on the power supply and verify that both gel box and power supply are working. Note: Black is negative, red is positive. (The DNA is negatively charged and will run towards the positive electrode.) Always Run to Red. 7. Run the gel at 80-150 V until the dye has migrated to an appropriate distance. Note: A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage. 8. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box. 9. Using any device that has UV light, visualize your DNA fragments. Note: The fragments of DNA are usually referred to as bands due to their appearance on the gel. 66
Viewing and Analyzing the Gel 1. Careful here: remember to wear gloves, and do not let the gel slip out of the tray! 2. Transfer the gel in its tray to the UV-viewing device. Slide the gel out of the tray and directly onto the UV table. 3. Turn on the UV. Remember to wear goggles! CAUTION: UV light is damaging to eyes and exposed skin. Protective eyewear should be worn at all times while using a UV light source. 67
4. Take a polaroid picture of your gel. 5. You can leave the used buffer in the gel tank for several more runs, just be sure to add to the buffer to completely submerge the gel. For best gel results, use completely fresh buffer. To clean: wash gel tray, lid, and tank with mild soap and warm water (not hot!) 6. Analyze the results with your supervisor. References 1. Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. Agarose Gel Electrophoresis for the Separation of DNA Fragments. J. Vis. Exp. (62), e3923, doi:10.3791/3923 (2012). 2. Joseph Sambrook and David W. Russel. Agarose Gel Electrophoresis 3. NS State University. Agarose Gel Electrophoresis Protocol (Making, Loading, Running, & Viewing) 4. Treseder Lab Protocol Molecular Techniques. Rev. 08/2007 5. Protocols in Molecular Biology (2000) 2.5A.1-2.5A.9. Contributed by Daniel Voytas Current Copyright 2000 by John Wiley & Sons, Inc. 6. www.lonza.com/research. Loading and Running DNA in Agarose gels 7. https://www.addgene.org/plasmidprotocols/gelelectrophoresis/ Good luck! Was this document helpful? Tell us on protocol_rcpn@outlook.com 68