Benchmarks DNA isolated by proteinase K digestion in molten agarose was used to visualize EBV replication intermediates on 2-dimensional agarose gels (data not shown). These structures, which exist in low numbers in cultured human Raji cells and have been detected elsewhere by others after extraction of 1.5 × 108 cells (10), could be detected with ≤7 × 106 cells—at least a 20-fold increase in sensitivity. Thus, digesting cells with proteinase K in molten agarose before plug formation is a rapid, less expensive and more efficient method for isolation of high-molecular-weight DNA. REFERENCES 1.Adams, A. and T. Lindahl. 1975. EpsteinBarr virus genomes with properties of circular DNA molecules in carrier cells. Proc. Natl. Acad. Sci. USA 72:1477-1481. 2.Applegate, M., G. Juhn, M. Liechty, M. Moore and J. Hozier. 1990. Use of DNA purified in situ from cells embedded in agarose plugs for the molecular analysis of tk-/- mutants recovered in the L5178Y tk± 3.7.2C mutagen assay system. Mutat. Res. 245:55-59. 3.Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl. 1998. Molecular Biology-Technique, Vol. 1. Greene Publishing, New York. 4.Birren, B. and E. Lai. 1993. Pulsed field gel electrophoresis: a practical guide. Academic Press, San Diego. 5.Gardiner, K. 1991. Pulsed field gel electrophoresis. Anal. Chem. 63:658-665. 6.Hatfull, G., A.T. Bankier, B.G. Barrell and P.J. Farrell. 1988. Sequence analysis of Raji Epstein-Barr virus DNA. Virology 164:334340. 7.Hirota, T., T. Kondoh, T. Matsumoto, Y. Jinno and N. Niikawa. 1989. Micro extraction of DNA from whole blood and amniocytes. Jinrui Idengaku Zasshi 34:217-223. 8.Johnson, P.G. and T.A. Beerman. 1994. Damage induced in episomal EBV DNA in Raji cells by antitumor drugs as measured by pulsed field gel electrophoresis. Anal. Biochem. 220:103-114. 9.Kuo, W.L., D.F. Deen, L.J. Marton and R. H. Shafer. 1987. Filter elution analysis of the effect of alpha-difluoromethylornithine on Xray-induced DNA damage in 9L cells. Radiat. Res. 109:68-77. 10.Little, R.D. and C.L. Schildkraut. 1995. Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin. Mol. Cell. Biol. 15:2893-2903. 11.McHugh, M.M., J.M. Woynarowski, L.S. Gawron, T. Otani and T.A. Beerman. 1995. Effects of the DNA-damaging enediyne C1027 on intracellular SV40 and genomic DNA in green monkey kidney BSC-1 cells. Biochemistry 34:1805-1814. 12.Okayasu, R. and G. Iliakis. 1992. The shape of DNA elution dose-response curves under 194 BioTechniques
non-denaturing conditions: the contribution of the degree of chromatin condensation. Int. J. Radiat. Biol. 61:455-463. 13.Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503517. 14.Yanamandra, G. and M.L. Lee. 1989. Isolation and characterization of human DNA in agarose block. Gene Anal. Tech. 6:71-74. 15.Yates, J., N. Warren, D. Reisman and B. Sugden. 1984. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc. Natl. Acad. Sci. USA 81:3806-3810.
This study was supported by grants from the National Cancer Institute (Grant Nos. CA 77491 and CA 16056) to T.A.B. Address correspondence to Dr. Mary M. McHugh, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Sts., Buffalo, NY 14263, USA. Internet:
[email protected] Received 3 August 1998; accepted 29 October 1998.
Mary M. McHugh and Terry A. Beerman Roswell Park Cancer Institute Buffalo, NY, USA
Plasmid Purification Using Hot Mg2+Treatment and No RNase BioTechniques 26:194-198 (February 1999)
Highest quality plasmid preparations can be obtained by centrifugation in an ethidium bromide/CsCl gradient (6). However, this method cannot be of routine use because it is expensive, time- and labor-consuming, produces toxic waste and requires high-speed centrifuges. Many alternative protocols have been proposed, and those using pancreatic RNase to degrade cellular RNA remain the most popular because of their simplicity and low cost. However, because of its high stability, RNase almost inevitably contaminates
the laboratory, which can be detrimental for experiments involving RNA. This necessitates the use of expensive placental RNase inhibitor every time RNA integrity is important (e.g., in cell-free transcription or translation reactions). Our experience shows that there is no need to add the inhibitor if RNase is not used. The key step of our protocol is degradation of RNA by heating in the presence of a high Mg2+ concentration rather than by use of RNase. We have optimized the conditions of the Mg2+ treatment to ensure preservation of the native structure of plasmid DNA, resulting in plasmid DNA that is indistinguishable from that prepared by the CsCl method. The procedure begins with a plasmid isolated by the alkaline lysis method of Birnboim and Doly (1) from 1.5 mL of overnight culture of Escherichia coli cells (Figure 1, lane 1). Unless otherwise stated, all incubations and centrifugations (at 12 000–16 000× g) were carried out in 1.5-mL microcentrifuge tubes. (i) Hot Mg2+ precipitation: the crude plasmid pellet (1) was resuspended in 80 µL of TE buffer (10 mM TrisHCl, pH 9.0, 1 mM EDTA), mixed with 10 µL of 1 M Tris-HCl, pH 9.0 and 10 µL of 1 M MgCl2, heated in a boiling water bath for 30 s, and cooled in an ice bath for another 30 s, and the precipitate was pelleted for 5 min. (ii) RNA hydrolysis: the supernatant was transferred into another tube and heated in a boiling water bath for 20 min. (iii) Cetyltrimethylammonium bromide (CTAB) precipitation: the sample was mixed with 100 µL of 0.2% CTAB in 200 mM NaCl, heated again for 30 s, chilled in an ice bath for 20 min and centrifuged for 10 min. The pellet was resuspended in 200 µL of 0.1% CTAB in 300 mM NaCl, spun down for 5 min and dissolved in 40 µL of 1.2 M NaCl in 50 mM Na-EDTA, pH 9.0. The plasmid was then precipitated with 100 µL of ethanol and washed with 70% ethanol, and the pellet was disolved in 20 µL of TE buffer (pH 9.0). It has been reported that most RNA precipitates together with chromosomal DNA from the alkaline plasmid preparation with 50 mM Mg2+ at room temperature (7). We confirmed this observation and found that when 100 mM Mg2+ was used, the precipitation was Vol. 26, No. 2 (1999)
Benchmarks more efficient, especially upon brief heating. Using these conditions, no appreciable loss of the covalently closed circular (CCC) plasmid was observed (Figure 1A, lanes 1–5). This treatment also precipitates most proteins. After ethanol-precipitation, the plasmid preparation obtained can be used in many genetic engineering procedures, including cloning, restriction analysis and cell transformation. The unprecipitated RNA is of tRNA size and is degraded upon 20 min of heating in the presence of 100 mM Mg2+, while the CCC plasmid remains intact (Figure 1A, lane 6) provided that the pH value of the Tris buffer at 20°C is 9.0. Heating at lower pH results in nicking and even fragmentation of the plasmid, whereas higher pH can denature the plasmid, resulting in both a change of its electrophoretic mobility and resistance to restriction endonucleases. Furthermore, we found that pH 9.0 preserves plasmid integrity better than pH 8.0, which is commonly used during other purification steps and storage. Large amounts of mono- and oligoribonucleotides present in the hydrolyzed sample can interfere with some applications such as plasmid sequenc-
ing. Their content can be reduced by repetitive ethanol-precipitation in the presence of 2 M ammonium acetate (5). However, a single precipitation of the plasmid with 0.1% CTAB (2,4) is more efficient. By introducing 32P-labeled RNA into the sample before the RNA hydrolysis step, we determined that the residual ribonucleotide content in the final plasmid preparation is less than 0.5% if the CTAB pellet is washed with 0.1% CTAB in 300 mM NaCl, and about 3% if washing is omitted. In separate experiments, we found that plasmid DNA is quantitatively precipitated by 0.1% CTAB if its concentration is at least 10 µg/mL and the NaCl concentration is between 50 and 300 mM. Using the higher salt helps to suppress the ionic binding of oligoribonucleotides to CTAB. Sometimes the hydrolyzed preparation contains aggregates visible at the top of the agarose gel after ethidium bromide staining (Figure 1A, lane 6). These aggregates do not interfere with most applications, including plasmid sequencing and transcription with T7 RNA polymerase; however, if desirable, they can be removed (Figure 1A, lane 7) by standard phenol extraction
(6) at plasmid concentrations of ≤100 µg/mL, which also eliminates any residual protein. The procedure is easily adapted to larger-scale purifications. Increasing the sample concentration 3-fold as compared with the small-scale protocol did not impair the yield or purity of the plasmid, which was the same as that of the control CsCl plasmid (Figure 1B). For a cell-culture volume of ≤200 mL, all incubations and centrifugations can be carried out in an appropriate number of microcentrifuge tubes. If larger tubes are used, the heating and cooling times should be increased to allow samples to equilibrate to the required temperature. Plasmids isolated by the described procedure are indistinguishable from those isolated by CsCl centrifugation in the following ways: (i) susceptibility to restriction endonuclease digestion (Figure 1B), (ii) performance in cell-free transcription in the absence of RNase inhibitor (Figure 1C), (iii) coupled transcription-translation, (iv) sequencing and (v) cell transformation (not shown). However, the procedure is as inexpensive, fast and simple as those using RNase. Its high reliability was confirmed in over 500 independent plasmid isolations with typical yields of 1–2 µg per 1 mL of cell culture. The procedure can also be used in other applications that require removal of RNA from DNA samples.
REFERENCES
Figure 1. Plasmid purification and properties. (A) Purification of plasmid pUC18 from transformed E. coli Z85 cells grown overnight in 1.5 mL of L-broth containing ampicillin at 50 µg/mL: lane 1, alkaline lysate; lane 2, pellet; lane 3, supernatant after precipitation with 100 mM Mg2+ at RT; lane 4, pellet; lane 5, supernatant after hot 100 mM Mg2+ precipitation; lanes 6 and 7, final preparations isolated without and with phenol extraction, respectively. (B) Large-scale pUC18 preparations isolated from 100 mL of culture using this method (lane 1), CsCl purification (lane 3) and the same preparations after XbaI digestion (lanes 2 and 4, respectively). Samples in Panels A and B were stained with ethidium bromide after electrophoresis through a 1% agarose gel (6); DNA in each lane was from 0.1 mL of cell culture. (C) Run-off transcripts (930 nucleotides) synthesized in the absence of RNase inhibitor by T7 RNA polymerase (3) from 5 ng of a plasmid isolated by this method (lane 1) or by CsCl purification (lane 2). Samples were stained with toluidine blue after electrophoresis through a 5% polyacrylamide gel in the presence of 7 M urea (6). 196 BioTechniques
1.Birnboim, H.C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 2.Del Sal, G., G. Manfioletti and C. Schneider. 1989. The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. BioTechniques 7:514-520. 3.Gurevich, V.V., I.D. Pokrovskaya, T.A. Obukhova and S.A. Zozulya. 1991. Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. Anal. Biochem. 195: 207-213. 4.Ishaq, M., B. Wolf and C. Ritter. 1990. Large-scale isolation of plasmid DNA using cetyltrimethylammonium bromide. BioTechniques 9:19-23. 5.Okayama, H. and P. Berg. 1982. High-efficiency cloning of full-length cDNA. Mol. Cell. Biol. 2:161-170. 6.Sambrook, J., E.F. Fritsch and T. Maniatis. Vol. 26, No. 2 (1999)
Benchmarks 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. CSH Laboratory Press, Cold Spring Harbor, NY. 7.Skovgaard, O. 1990. Selective precipitation of RNA with Mg2+ improves the purification of plasmid DNA. Trends Genet. 6:140.
We thank Drs. A.P. Alimov, A.A. Demidenko, I.V. Kolesnikov and P.N. Simonenko for help in evaluating plasmid quality and reproducibility of the method. The work was supported by Grant Nos. 96-04-48329 (to H.V.C.) and 96-04-48331 from the Russian Foundation for Basic Research (RFBR), and the International Association for the promotion of cooperation with scientists from the New Independent States of the former Soviet Union (INTAS)-RFBR Grant No. 95-1365, and an International Research Scholar's award from the Howard Hughes Medical Institute to A.B.C. Address correspondence to Dr. Alexander B. Chetverin, Institute of Protein Research, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia. Internet: alexch@vega. protres.ru Received 3 August 1998; accepted 19 October 1998.
Victor I. Ugarov, Timur R. Samatov, Helena V. Chetverina and Alexander B. Chetverin Institute of Protein Research Russian Academy of Sciences Pushchino, Moscow Region, Russia
archival tissues to determining genetic abnormalities in malignancies such as lymphomas (4). The first step in processing tissue sections usually involves deparaffinization to remove the paraffin from the tissue by dissolving it in an appropriate solvent such as octane. Using one standard protocol, this process can take 1–2 h to complete and requires multiple centrifugation steps (5). Additionally, tissue may be lost after each round of centrifugation in octane because the tissue fragments tend to float away from the pellet, especially when wearing latex gloves, which create electrostatic forces that can disperse the tissue pellet. We have found the standard deparaffinization protocol to be cumbersome and time-consuming. Others have tried to improve the deparaffinization process by using methods such as microwave treatment (1) and boiling (7) or heating (9) of samples to melt the paraffin so that it can be separated from tissue and removed. However, the high temperatures used in these methods could damage DNA. Others have tried to bypass the deparaffinization step altogether (3). A novel modification to an existing protocol was designed to make the deparaffinization process simpler and faster and to avoid the use of heat.
In the standard deparaffinization protocol (10), 1.0 mL of octane is added to the tissue section in a 1.5-mL microcentrifuge tube, and the tube is mixed at room temperature for 30 min. The tissue is pelleted by centrifugation for 3–5 min at 16 000× g, and the octane is removed with a pipet. This process is repeated by adding another 1.0 mL of octane, followed by mixing for 30 min, centrifugation at 16 000× g and removal of octane. A 0.5-mL vol of 100% ethanol is then added to the tube to wash away the residual octane and is mixed by inversion. The tissue is centrifuged, and the ethanol is removed with a pipet. Another 0.5-mL vol of ethanol is added, mixed by inversion and centrifuged again at 16 000× g. After removing the ethanol from the tissue pellet, the sample is dried by vacuum. Our modified protocol is described in Table 1. We have found that a single 25-µm tissue section is completely deparaffinized after being vortex mixed for 10 s in 1.0 mL of octane. Unlike ethanol, methanol is not miscible in octane and separates in the tube along with the tissue fragments. Thus, in our rapid protocol, the methanol serves to cleanly separate the tissue from the octane so that the octane can be completely removed.
Paraffin Removal from Tissue Sections for Digestion and PCR Analysis BioTechniques 26:198-200 (February 1999)
Amplification of DNA from fixed, paraffin-embedded tissues using the polymerase chain reaction (PCR) has many applications, from detecting microbes such as human papilloma virus (8) and human herpesvirus 8 (6) in 198 BioTechniques
Figure 1. Agarose gel of amplification products from a β-globin PCR. The product size is 268 bp. Lanes 1 and 2, lymph node tissue deparaffinized with method A (rapid) and digested (duplicate samples). Lanes 3 and 4, lymph node tissue deparaffinized with method B (standard) and digested (duplicate samples). Lane 5, reagent only control. Lane 6, digestion buffer control. Lane 7, human lymphocyte DNA positive control. Lane 8, 1-kb DNA ladder (Life Technologies, Gaithersburg, MD, USA), arrow indicates a 298-bp band in the ladder. Vol. 26, No. 2 (1999)
