Science, Tech, Math › Science How to Make 10X TBE Electrophoresis Buffer Share Flipboard Email Print Cl4ss1cr0ck3R / Creative Commons Science Chemistry Basics Chemical Laws Molecules Periodic Table Projects & Experiments Scientific Method Biochemistry Physical Chemistry Medical Chemistry Chemistry In Everyday Life Famous Chemists Activities for Kids Abbreviations & Acronyms Biology Physics Geology Astronomy Weather & Climate By Anne Marie Helmenstine, Ph.D. Chemistry Expert Ph.D., Biomedical Sciences, University of Tennessee at Knoxville B.A., Physics and Mathematics, Hastings College Dr. Helmenstine holds a Ph.D. in biomedical sciences and is a science writer, educator, and consultant. She has taught science courses at the high school, college, and graduate levels. our editorial process Facebook Facebook Twitter Twitter Anne Marie Helmenstine, Ph.D. Updated July 12, 2019 TBE and TAE are used as buffers in molecular biology, primarily for electrophoresis of nucleic acids. Tris buffers are used under slightly basic pH conditions, as for DNA electrophoresis, because this keeps the DNA soluble in the solution and deprotonated so it will be attracted to the positive electrode and will migrate through a gel. EDTA is an ingredient in the solution because this common chelating agent protects nucleic acids from degradation by enzymes. The EDTA chelates divalent cations that are cofactors for nucleases that may contaminate the sample. However, since the magnesium cation is a cofactor for DNA polymerase and restriction enzymes, the concentration of EDTA is kept purposely low (around 1 mM concentration). 10X TBE Electrophoresis Buffer Materials 108 g of Tris base [tris(hydroxymethyl)aminomethane]55 g of boric acid7.5 g of EDTA, disodium saltDeionized water Preparation for the 10X TBE Electrophoresis Buffer Dissolve the Tris, boric acid, and EDTA in 800 ml of deionized water.Dilute the buffer to 1 L. Undissolved white clumps may be made to dissolve by placing the bottle of solution in a hot water bath. A magnetic stir bar can aid the process. You do not need to sterilize the solution. Although precipitation may occur after a span of time, the stock solution is still usable. You can adjust the pH using a pH meter and dropwise addition of concentrated hydrochloric acid (HCl). It's fine to store TBE buffer at room temperature, although you may wish to filter the stock solution through a 0.22-micron filter to remove particle that would foster precipitation. 10X TBE Electrophoresis Buffer Storage Store the bottle of 10X buffer solution at room temperature. Refrigeration will accelerate precipitation. Using 10X TBE Electrophoresis Buffer The solution is diluted before use. Dilute 100 mL of 10X stock to 1 L with deionized water. 5X TBE Stock Solution Recipe The advantage of the 5X solution is that it's less likely to precipitate. 54 g of Tris base (Trizma)27.5 grams of boric acid20 mL of 0.5 M EDTA solutionDeionized water Preparation Dissolve the Tris base and boric acid in the EDTA solution.Adjust the pH of the solution to 8.3 using concentrated HCl.Dilute the solution with deionized water to make 1 liter of 5X stock solution. The solution may also be diluted to 1X or 0.5X for electrophoresis. Using a 5X or 10X stock solution by accident will give you poor results because as much heat will be generated. In addition to giving you poor resolution, the sample may be damaged. 0.5X TBA Buffer Recipe 5X TBE stock solutionDistilled deionized water Preparation Add 100 mL of the 5X TBE solution to 900 mL of distilled deionized water. Mix thoroughly before use. Limitations Although TBE and TAE are common electrophoresis buffers, there are other options for low-molarity conductive solutions, including lithium borate buffer and sodium borate buffer. The problem with TBE and TAE are that Tris-based buffers limit the electric field that can be used in electrophoresis because too much charge causes a runaway temperature.