Running DNA Mini-Gels in 20 Minutes or Less Using Sodium Boric Acid Buffer

Abstract

Providing a biotechnology experience for students can be challenging on several levels, with time becoming a real constraint in many experiments. Many techniques for DNA isolation and analysis involve a gel electrophoresis step that usually requires a large block of time. In addition to the time needed for loading the samples, which can be substantial for novices, DNA gels typically require 45 minutes to run using standard TBE (Tris-boric acid-disodium EDTA) or TAE (Tris-acetic acid-disodium EDTA) buffers. In a 50-minute class period it is virtually impossible for students to load samples, run and analyze their gels. Therefore any procedure involving gel electrophoresis becomes a challenge. At the BioPharmaceutical Technology Center Institute (BTCI) we of[er a variety of biotechnology field trips including a polymerase chain reaction (PCR) and a restriction enzyme digest, where we are often pressed for time both running and analyzing gels. Recently a paper was published (Brody & Kern, 2004) in which a sodium boric acid buffer was used to run gels much faster without loss of resolution. We tested this sodium boric acid buffer in our field trip program and were able to reduce the time required to run gels from 45 minutes to 20 minutes or less. This sodium boric acid buffer can be made relatively easily with common chemicals, or the stock solution can be ordered (Faster Better Media). Materials Chemicals NaOH/sodium hydroxide (Mallinkrodt Cat#7708) [H.sub.3]B[O.sub.3] /boric acid (FisherBiotech BP168-500, electrophoresis grade) 0.5X TBE, agarose (Promega, cat#V3121) ethidium bromide (Promega, cat#HS041) 6X loading dye (Promega cat#G1881) lambda DNA HindIII/EcoRI molecular weight markers (Promega cat#G1731) 5X SB[TM] loading medium (Faster Better Media cat #SB5N-8) 1XSB Hi-Lo DNA Ladder (Faster Better Media) EcoRI (Promega cat#R6011) HindIII (Promega cat#R6041) PstL (Promega cat#R6111) Safety Note: Ethidium bromide can detect smaller quantities of DNA than other DNA dyes because of its high affinity for DNA. However, that high affinity for DNA makes ethidium bromide a strong mutagen, and possibly a teratogen and carcinogen. In addition, a UV light source is required to detect DNA stained with ethidium bromide. UV light can damage skin and eyes. As a consequence of these two potential health hazards, many school districts do not allow the use of ethidium bromide in classrooms. In districts where ethidium bromide use is permitted, safety and disposal protocols vary. Teachers should check with their school districts before using ethidium bromide. There are many other dyes for detection of DNA, however the more common dyes require much larger quantities of DNA for visualization and shorter pieces of DNA may be difficult to visualize at all. Carolina Blu (Carolina Biological Supply Company) worked as well in sodium borate as in TBE buffer when tested in our lab, however it is three to four times less sensitive than ethidium bromide and requires at least an hour to stain and destain before samples can be viewed. SYBR Gold (Invitrogen) and GelStar (Cambrex) stains are sensitive and both work with sodium borate (Kern, personal communication), however they require UV transilluminators for detection. Other dyes may vary in their effectiveness with the sodium borate system, thus teachers are encouraged to test other dyes prior to use in class. Equipment Stir plate pH meter (for making buffer stock, not necessary if ordering buffer) Electrophoresis Equipment FOTO/Force 250 power supply (cat#ET-4297) a high voltage FOTODYNE power supply and minisingle ceil electrophoresis chamber (cat# E1-1408) FOTO/Phoresis UV transilluminator (Model 1-1430) FOTODYNE Polaroid camera (cat# 1-1440) from FOTODYNE Inc., 950 Walnut Ridge Drive, Hartland WI 53029; Tel. …