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10.1021/ed100237e Published on Web 07/30/2010

In the Laboratory

An Inexpensive, Relatively Green, and RapidMethod To Purify Genomic DNA from Escherichia

coli: An Experiment for the UndergraduateBiochemistry LaboratoryPaul A. Sims,* Katie M. Branscum, and Lydia KaoDepartment of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019*psims@ou.edu

Virginia R. KeavenyDepartment of Chemistry, Minot State University, Minot, North Dakota 58707

The purification of genomic DNA is a desirable andimportant activity in the modern undergraduate biochemistrylaboratory. Purified DNA can serve as the starting point for aseries of subsequent investigations, including the amplification ofa particular gene within the DNA via the polymerase chainreaction (PCR). To develop a method of purifying genomicDNA that was suitable for this use in the undergraduatelaboratory, we considered the following criteria: (i) the cost ofthe 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 areavailable from commercial suppliers because of concerns aboutcost (∼$2.00/preparation with many commercial kits versus∼$0.20/preparation with the method reported herein) andbecause many of the protocols associated with the kits tooktoo long to perform.2 Therefore, we searched the literature andconsidered a number of published protocols. One protocol fromthis 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 ofthe environmental effects of these substances. Other publishedprotocols were less time-consuming, but most of these also usedchloroform, phenol, and isoamyl alcohol. In fact, none of thepublished protocols completely met our criteria, but two pub-lished protocols seemed to be more rapid and greener than any ofthe 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 havefewer hazards associated with their use and disposal (see forexample, ref 4), we decided that this method would be greeneroverall. When we tried this method, however, we met withlimited 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 detergentsPersil Mega Perls or Frosch were used to help lyse the cells followedby an extraction with phenol/chloroform/isoamylalcohol (25:24:1),treatment with ribonuclease (RNase), and finally another extrac-tion but with chloroform/isoamylalcohol. The authors of thissecond study reasoned that commercial laundry detergents oftencontain, in addition to detergents, various hydrolytic enzymes such asproteases and lipases and ethylenediaminetetraacetic acid (EDTA).

The detergent and lipases help break down cell membranes; theproteases help degrade cellular proteins including (possibly) deoxyri-bonucleases (DNases), whichwould catalyze the undesirable cleavageof DNA; and the EDTA chelates metal ions, which are required forDNase activity. We reasoned that a combination of the above twoprotocols would mostly satisfy our three criteria and allow us todevelop a protocol that could be completed in less than∼2 h so thatthe remaining hour (in a typical 3-h undergraduate laboratory) couldbe devoted to the preparation of a PCR in which the purifiedgenomic DNA is used as the template DNA.

A separation scheme highlighting the overall method isshown in Figure 1, and a condensed version of the protocol

edited byMary M. Kirchhoff

American Chemical SocietyWashington, DC 20036

Figure 1. Separation scheme showing the steps used to isolate genomicDNA of Escherichia coli.

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(suitable for following in the laboratory) is presented in thesupporting information. Students in the introductory bio-chemical class are given the condensed version and are expectedto draw their own separation scheme as part of the prelaboratoryexercise. A brief description of the method is provided below, buta more detailed description of the method, including severalexplanatory side notes and possible variations, is included in thesupporting information.

Methods and Materials

A 1-1.4 mL aliquot of an overnight culture of E. coliK12 isadded to a 1.5 mL microcentrifuge tube, and the sample is spunfor 1 min at top speed (∼16,000g) in a microcentrifuge such thatthe 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 arethoroughly resuspended in the remaining (0.4 mL) of super-natant. Tide Free 2� Ultra, 5 μL, which contains enzymes butdoes not contain extraneous dyes or perfumes, is added to theresuspended cells. The mixture is vortexed briefly (three 2-spulses) and then held at 37 �C for 10 min. After the 37 �Cincubation, 0.75 mL of n-butanol is added to the resuspendedcells, and the tube is vortexed briefly (two 5-s pulses). Themixture is then spun at top speed for 5 min.

The genomicDNA is removed by carefully inserting a sterile10-200 μL pipet tip (attached to a pipet) through the organiclayer and into the lower aqueous layer. As the tip is inserted, itlikely will be necessary to push aside the film of denatured proteinat the interface of the two layers. Then, 200 μL of the aqueous

layer is slowly removed, taking care not to disturb the pellet at thebottom of the tube or to siphon any of the material at theinterface (Figure 2B).

The withdrawn aqueous layer is transferred to a sterilemicrocentrifuge tube, and 700 μL of 2-propanol is slowly addedfollowed by gentle mixing in which the tube is slowly inverted∼5times. The sample is spun at top speed for 5 min, and afterward,the supernatant is carefully decanted into the waste beaker andthe tube is drained onto a Kim-wipe or other suitable absorbent.A pellet should be visible toward the back, bottom portion of thetube (Figure 2C). Next, 700 μL of 70% ethanol is added to thetube, which is mixed by gentle inversion, followed by a spin at topspeed for 2 min. After this last spin, the supernatant is carefullyremoved by siphoning with a drawn-out Pasteur pipet, whichshould be inserted along the “belly” of the tube so as not disturbthe pellet (Figure 2C).

The open tube is allowed to air-dry for∼5 min, after whichtime it is checked to ensure that no visible traces of liquid arepresent. When the sample appears dry, 50 μL of a solutioncontaining 0.1 mg/mL RNase A in autoclaved distilled H2O isadded, and the sample is incubated at 60 �C for 60 min. Midwaythrough the incubation, the tube should be removed and gentlytapped to dislodge the pellet from the side or bottom of the tubeand to begin bringing the pellet into solution. After the incuba-tion, a 1 μL aliquot of sample can be used as template DNA in aPCR.

Utility of the Method

We have used the above method with good success in theundergraduate laboratory as part of the first step in a series ofrelated laboratory investigations in which the students amplify agene, clone the gene into an expression vector, and ultimatelypurify and assay the corresponding protein product of the gene.The gene that is amplified in this sequence of laboratoryinvestigations is an alcohol dehydrogenase. To determine if theprotocol provided a suitable template for the amplification ofother genes in E. coli, we ran several PCRs designed to amplifyvarious genes from different regions of the genome as indicatedin Figure 3A.3 The gel shown in Figure 3B indicates that theisolated genomic DNA was a suitable template for the amplifica-tion of these other genes as the positions of the bands within thegel are consistent with the respective sizes of the various genes.Thus the method provides genomic DNA that appears to bereasonably representative of the E. coli genome.

The PCR results are consistent with the observations of theappearance of the isolated genomic DNA in a 0.8% agarose gel.Much of the isolated genomic DNA runs essentially as a singleband, and the position of this band is above the position of thehighest molecular weight marker (10 kb) in the adjacent lane ofthe gel (Figure 3B; lanes 1 and 2); however, some of the isolatedgenomic DNA is so large that it does not migrate in the gel andinstead remains in the well (Figure 3B; lane 2). A faint, diffuseband of degraded RNA also can be seen toward the bottom of thegel in this lane.

Hazards

Although the K12 strain of E. coli is nonpathogenic, thestudents should observe all standard laboratory precautions (e.g.,safety goggles should be worn at all times; gloves should be worn

Figure 2. (A) Top viewof amicrocentrifuge rotor andan expanded viewof the positioning of themicrocentrifuge tube in the rotor such that the caphinge faces outward. (B) Side view of a microcentrifuge tube as it shouldappear after the addition of n-butanol and the subsequent centrifugation.Note that the pipet tip is positioned to withdraw the aqueous phasewithout disturbing the pellet of cellular debris; the interface film has beenpushed aside by the pipet tip. (C) Side view of a microcentrifuge tubeshowing how the supernatant of 70% ethanol is to be siphoned with adrawn-out Pasteur pipet.

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when handling the bacterial culture; biohazard waste should betreated with bleach; pipet tips and microcentrifuge tubes that werein contact with the bacterial culture should be placed in biohazardbags and autoclaved; care should be taken inhandling the drawn-outPasteur pipet). Ribonuclease A is irritating to the eyes, skin, andrespiratory system. The solvents n-butanol, 2-propanol, and ethanolare flammable and harmful if swallowed, inhaled, or absorbedthrough skin. The MSDS of each of these solvents should beconsulted by the students prior to starting the laboratory, and theappropriate disposal guidelines should be followed.

Summary

A method was presented that allows undergraduate stu-dents, even in large laboratory classes, to isolate genomic DNAfrom E. coli in a manner that is economical and relatively greenand rapid. These features were attained in part by the use of thelaundry detergent Tide Free 2� Ultra in place of SDS, and thesolvent n-butanol in place of the more commonly used chloro-form, isoamylalcohol, and phenol. Using the method describedherein, undergraduate students can isolate genomic DNA ofE. coli and use this genomic DNA as a template in a PCR, whichcan be set up the same laboratory period.

Acknowledgment

The author gratefully acknowledges Paul F. Cook for help-ful comments and suggestions. The author also is grateful toVidya Kumar for helpful comments and suggestions concerningthe implementation of this activity in large undergraduatelaboratory classes.

Notes1. Although “greenness” encompasses many aspects such as atom

economy, sustainability, toxicity, and so forth, our focus herein

is on the substitution of hazardous solvents with less-hazardousones, which is consistent with one of the goals of greenchemistry (1).

2. At least one kit-based method was projected to take less timethan the method reported herein (1 h versus 1.7 h), but the costwas somewhat higher at ∼$2.30/preparation.

3. The relative positions of the genes shown in Figure 3A werefound in the BioCyc database (6) using the sequence of E. coliK12 as determined by Blattner et al. (7).

Literature Cited

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; studentinstructions with a condensed protocol (suitable for following in thelaboratory) and some pre- and postlaboratory questions and answers.This material is available via the Internet at http://pubs.acs.org.

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 toamplify 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 containsgene 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 containsgene g.