In the Laboratory edited by
Mary M. Kirchhoff American Chemical Society Washington, DC 20036
An Inexpensive, Relatively Green, and Rapid Method To Purify Genomic DNA from Escherichia coli: An Experiment for the Undergraduate Biochemistry Laboratory Paul A. Sims,* Katie M. Branscum, and Lydia Kao Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019 *
[email protected] Virginia R. Keaveny Department of Chemistry, Minot State University, Minot, North Dakota 58707
The purification of genomic DNA is a desirable and important activity in the modern undergraduate biochemistry laboratory. Purified DNA can serve as the starting point for a series of subsequent investigations, including the amplification of a particular gene within the DNA via the polymerase chain reaction (PCR). To develop a method of purifying genomic DNA that was suitable for this use in the undergraduate laboratory, we considered the following criteria: (i) the cost of the method; (ii) length of time to complete the method; and (iii) the “greenness” of the method (1).1 We opted not to use any of the various “kits” that are available from commercial suppliers because of concerns about cost (∼$2.00/preparation with many commercial kits versus ∼$0.20/preparation with the method reported herein) and because many of the protocols associated with the kits took too long to perform.2 Therefore, we searched the literature and considered a number of published protocols. One protocol from this Journal (2) was thorough, but it was somewhat lengthy (>10 h to complete) and it used sodium perchlorate, chloroform, isoamylalcohol, and phenol, which we hoped to avoid because of the environmental effects of these substances. Other published protocols were less time-consuming, but most of these also used chloroform, phenol, and isoamyl alcohol. In fact, none of the published protocols completely met our criteria, but two published protocols seemed to be more rapid and greener than any of the other reported methods. In the first of these two protocols (3), n-butanol or a combination of n-butanol and 0.1% (m/v) sodium dodecyl sulfate (SDS) was used, along with 70% (v/v) ethanol “washes”. Because both n-butanol and ethanol have fewer hazards associated with their use and disposal (see for example, ref 4), we decided that this method would be greener overall. When we tried this method, however, we met with limited success, as judged by the lack of DNA band(s) in the 0.8% (m/v) agarose gels that were used to check the results. In the second of the two protocols (5), the laundry detergents Persil Mega Perls or Frosch were used to help lyse the cells followed by an extraction with phenol/chloroform/isoamylalcohol (25:24:1), treatment with ribonuclease (RNase), and finally another extraction but with chloroform/isoamylalcohol. The authors of this second study reasoned that commercial laundry detergents often contain, in addition to detergents, various hydrolytic enzymes such as proteases and lipases and ethylenediaminetetraacetic acid (EDTA).
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Figure 1. Separation scheme showing the steps used to isolate genomic DNA of Escherichia coli.
The detergent and lipases help break down cell membranes; the proteases help degrade cellular proteins including (possibly) deoxyribonucleases (DNases), which would catalyze the undesirable cleavage of DNA; and the EDTA chelates metal ions, which are required for DNase activity. We reasoned that a combination of the above two protocols would mostly satisfy our three criteria and allow us to develop a protocol that could be completed in less than ∼2 h so that the remaining hour (in a typical 3-h undergraduate laboratory) could be devoted to the preparation of a PCR in which the purified genomic DNA is used as the template DNA. A separation scheme highlighting the overall method is shown in Figure 1, and a condensed version of the protocol
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r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 87 No. 10 October 2010 10.1021/ed100237e Published on Web 07/30/2010
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In the Laboratory
Figure 2. (A) Top view of a microcentrifuge rotor and an expanded view of the positioning of the microcentrifuge tube in the rotor such that the cap hinge faces outward. (B) Side view of a microcentrifuge tube as it should appear after the addition of n-butanol and the subsequent centrifugation. Note that the pipet tip is positioned to withdraw the aqueous phase without disturbing the pellet of cellular debris; the interface film has been pushed aside by the pipet tip. (C) Side view of a microcentrifuge tube showing how the supernatant of 70% ethanol is to be siphoned with a drawn-out Pasteur pipet.
(suitable for following in the laboratory) is presented in the supporting information. Students in the introductory biochemical class are given the condensed version and are expected to draw their own separation scheme as part of the prelaboratory exercise. A brief description of the method is provided below, but a more detailed description of the method, including several explanatory side notes and possible variations, is included in the supporting information. Methods and Materials A 1-1.4 mL aliquot of an overnight culture of E. coli K12 is added to a 1.5 mL microcentrifuge tube, and the sample is spun for 1 min at top speed (∼16,000g) in a microcentrifuge such that the hinges connecting the caps to the tubes are oriented outward, which facilitates locating the pellets (Figure 2). After spinning, 0.6-1.0 mL of supernatant is removed, and the pelleted cells are thoroughly resuspended in the remaining (0.4 mL) of supernatant. Tide Free 2 Ultra, 5 μL, which contains enzymes but does not contain extraneous dyes or perfumes, is added to the resuspended cells. The mixture is vortexed briefly (three 2-s pulses) and then held at 37 °C for 10 min. After the 37 °C incubation, 0.75 mL of n-butanol is added to the resuspended cells, and the tube is vortexed briefly (two 5-s pulses). The mixture is then spun at top speed for 5 min. The genomic DNA is removed by carefully inserting a sterile 10-200 μL pipet tip (attached to a pipet) through the organic layer and into the lower aqueous layer. As the tip is inserted, it likely will be necessary to push aside the film of denatured protein at the interface of the two layers. Then, 200 μL of the aqueous 1114
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layer is slowly removed, taking care not to disturb the pellet at the bottom of the tube or to siphon any of the material at the interface (Figure 2B). The withdrawn aqueous layer is transferred to a sterile microcentrifuge tube, and 700 μL of 2-propanol is slowly added followed by gentle mixing in which the tube is slowly inverted ∼5 times. The sample is spun at top speed for 5 min, and afterward, the supernatant is carefully decanted into the waste beaker and the tube is drained onto a Kim-wipe or other suitable absorbent. A pellet should be visible toward the back, bottom portion of the tube (Figure 2C). Next, 700 μL of 70% ethanol is added to the tube, which is mixed by gentle inversion, followed by a spin at top speed for 2 min. After this last spin, the supernatant is carefully removed by siphoning with a drawn-out Pasteur pipet, which should be inserted along the “belly” of the tube so as not disturb the pellet (Figure 2C). The open tube is allowed to air-dry for ∼5 min, after which time it is checked to ensure that no visible traces of liquid are present. When the sample appears dry, 50 μL of a solution containing 0.1 mg/mL RNase A in autoclaved distilled H2O is added, and the sample is incubated at 60 °C for 60 min. Midway through the incubation, the tube should be removed and gently tapped to dislodge the pellet from the side or bottom of the tube and to begin bringing the pellet into solution. After the incubation, a 1 μL aliquot of sample can be used as template DNA in a PCR. Utility of the Method We have used the above method with good success in the undergraduate laboratory as part of the first step in a series of related laboratory investigations in which the students amplify a gene, clone the gene into an expression vector, and ultimately purify and assay the corresponding protein product of the gene. The gene that is amplified in this sequence of laboratory investigations is an alcohol dehydrogenase. To determine if the protocol provided a suitable template for the amplification of other genes in E. coli, we ran several PCRs designed to amplify various genes from different regions of the genome as indicated in Figure 3A.3 The gel shown in Figure 3B indicates that the isolated genomic DNA was a suitable template for the amplification of these other genes as the positions of the bands within the gel are consistent with the respective sizes of the various genes. Thus the method provides genomic DNA that appears to be reasonably representative of the E. coli genome. The PCR results are consistent with the observations of the appearance of the isolated genomic DNA in a 0.8% agarose gel. Much of the isolated genomic DNA runs essentially as a single band, and the position of this band is above the position of the highest molecular weight marker (10 kb) in the adjacent lane of the gel (Figure 3B; lanes 1 and 2); however, some of the isolated genomic DNA is so large that it does not migrate in the gel and instead remains in the well (Figure 3B; lane 2). A faint, diffuse band of degraded RNA also can be seen toward the bottom of the gel in this lane. Hazards Although the K12 strain of E. coli is nonpathogenic, the students should observe all standard laboratory precautions (e.g., safety goggles should be worn at all times; gloves should be worn
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In the Laboratory
Figure 3. (A) Relative positions of genes a-h on the circular E. coli genome. (B) A 0.8% agarose gel in which samples from PCRs designed to amplify genes a-h were run. Lane 1 contains a 1 kb DNA ladder; lane 2 contains the isolated genomic DNA; lane 3 contains gene c; lane 4 contains gene a; lane 5 contains gene h; lane 6 contains gene d; lane 7 contains gene b; lane 8 contains gene f; lane 9 contains gene e; and lane 10 contains gene g.
when handling the bacterial culture; biohazard waste should be treated with bleach; pipet tips and microcentrifuge tubes that were in contact with the bacterial culture should be placed in biohazard bags and autoclaved; care should be taken in handling the drawn-out Pasteur pipet). Ribonuclease A is irritating to the eyes, skin, and respiratory system. The solvents n-butanol, 2-propanol, and ethanol are flammable and harmful if swallowed, inhaled, or absorbed through skin. The MSDS of each of these solvents should be consulted by the students prior to starting the laboratory, and the appropriate disposal guidelines should be followed. Summary
Literature Cited
A method was presented that allows undergraduate students, even in large laboratory classes, to isolate genomic DNA from E. coli in a manner that is economical and relatively green and rapid. These features were attained in part by the use of the laundry detergent Tide Free 2 Ultra in place of SDS, and the solvent n-butanol in place of the more commonly used chloroform, isoamylalcohol, and phenol. Using the method described herein, undergraduate students can isolate genomic DNA of E. coli and use this genomic DNA as a template in a PCR, which can be set up the same laboratory period. Acknowledgment The author gratefully acknowledges Paul F. Cook for helpful comments and suggestions. The author also is grateful to Vidya Kumar for helpful comments and suggestions concerning the implementation of this activity in large undergraduate laboratory classes. Notes
1. Although “greenness” encompasses many aspects such as atom economy, sustainability, toxicity, and so forth, our focus herein
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is on the substitution of hazardous solvents with less-hazardous ones, which is consistent with one of the goals of green chemistry (1). 2. At least one kit-based method was projected to take less time than the method reported herein (1 h versus 1.7 h), but the cost was somewhat higher at ∼$2.30/preparation. 3. The relative positions of the genes shown in Figure 3A were found in the BioCyc database (6) using the sequence of E. coli K12 as determined by Blattner et al. (7).
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1. Doxsee, K. M.; Hutchison, J. E. Green Organic Chemistry, 1st ed.; Thomson Brooks/Cole: Toronto, 2004; Chapter 6; p 23. 2. Wilson, W. D.; Davidson, M. W. J. Chem. Educ. 1979, 56, 204–206. 3. Mak, Y. M.; Ho, K. K. Nucleic Acids Res. 1992, 20, 4101–4102. 4. Guidelines for drain disposal of chemicals. http://www.ehs.berkeley. edu/pubs/guidelines/draindispgls.html (accessed Jul 2010). 5. Bahl, A.; Pfenninger, M. Nucleic Acids Res. 1996, 24, 1587–1588. 6. Keseler, I. M.; Collado-Vides, J.; Gama-Castro, S.; Ingraham, J.; Paley, S.; Paulsen, I. T.; Peralta-Gil, M.; Karp, P. D. Nucleic Acids Res. 2005, 33, D334–D337. 7. Blattner, F. R.; Plunkett, G., III; Bloch, C. A.; Perna, N. T.; Burland, V.; Riley, M.; Collado-Vides, J.; Glasner, J. D.; Rode, C. K.; Mayhew, G. F.; Gregor, J.; Davis, N. W.; Kirkpatrick, H. A.; Goeden, M. A.; Rose, D. J.; Mau, B.; Shao, Y. Science 1997, 277, 1453–1474.
Supporting Information Available An expanded version of the Materials and Methods section; instructor notes and a list of required equipment and chemicals; student instructions with a condensed protocol (suitable for following in the laboratory) and some pre- and postlaboratory questions and answers. This material is available via the Internet at http://pubs.acs.org.
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