Paraffin wax as a vapor barrier for the PCR.

Abstract

Department of Pathology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9072 The PCR is a highly efficient method for amplification of specific DNA sequences. (1) The use of thermostable DNA polymerases allows reaction to proceed after repeated exposures to elevated temperatures that are required for strand separation. (z) To prevent evaporation of reaction at these elevated temperatures, reaction is overlaid with mineral oil to form a vapor barrier. Mineral oil, also known as paraffin oil, is a liquid at room temperature and requires post-PCR chloroform extractions for its removal. We describe use of reagent-grade paraffin wax as a vapor barrier for PCR reactions in place of more costly commercial paraffin wax preparations that are marketed specifically as vapor barriers for PCR. Paraffin wax allows separation of reaction components prior to heating and upon cooling solidifies to allow easy removal of reaction mixture from tube without need for chloroform extractions. The paraffin can also be melted again to form a barrier for prolonged storage at 4~ PCR reactions were carried out in a total volume of 100 Izl and consisted of 50 mM KCI, 10 mM Tris-HC1 (pH 8.8), 1.5 mM MgCl,z 0.1% Triton X-100, 0.2 mM dNTPs, 1.3 ~M of each primer, and 50 ~l of mineral oil (Cat. No. M5904, Sigma) or 50 pLl of melted paraffin wax. The template DNA, a pGEM3 plasmid containing a 1-kb insert, was isolated from Escherichia coli HB101 by centrifuging 0.1 ml of an overnight culture, resuspending pellet in original volume of 0.1% Tween 20, and then boiling for 10 min. Cellular debris was pelleted by centrifugation for 5 min in a microfuge, and 3 ~l of supernatant was added to each reaction. The primers used were complementary to T7 (5'-AATACGACTCACTATAG-3') and SP6 (5'-ATTTAGGTGACACTATAG-3') promoters. The reactions were thermally denatured at 85~ for 5 m i n and cooled to 25~ to allow wax to solidify; 1.5 units of Taq DNA polymerase was added directly to reaction that used mineral oil or was added directly onto the solidified wax. The reactions were conducted according to touchdown PCR procedure, (3) where primers are annealed for 1 m i n at 52~ for first cycle, then decreased 1~ each cycle unti l reaching 42~ The PCR was allowed to proceed for 40 cycles, then held at 4~ The wax solidified allowing reaction to be removed by punch ing through wax layer with pipette tip and removing reaction mixture. The reaction with mineral oil was extracted twice with chloroform. The products were analyzed by agarose gel electrophoresis. (4) We tested several types of paraffin wax: Aldrich (Cat. No. 32,721-2, m.p. 5963~ Surgipath (Cat. No. EM400, m.p. 55-57~ and Baxter (Cat. No. M73461A, m.p. 55-57~ All paraffin waxes tested worked as a vapor barrier, but Aldrich paraffin proved superior as it provided most product with least background (Fig. 1). The Aldrich paraffin was a reagent-grade paraffin that contained no additives. The other two paraffins were for tissue embedding and contained additives. Also, Aldrich paraffin had a slightly higher mel t ing point, which allowed reaction to reach a higher temperature before melting and allowing Taq DNA polymerase to enter reaction. The use of wax as a vapor barrier provides two significant advantages over mineral oil: (1) no extractions with organic solvents are necessary, and (2) reaction components