PROTOCOL

A reliable general purpose method for extracting genomic DNA from Dictyostelium cells
Karen E Pilcher1, Petra Fey1, Pascale Gaudet1, Anthony S Kowal2, & Rex L Chisholm1,2
1dictyBase,

Center for Genetic Medicine, Northwestern University, 676 North Saint Clair Street Suite 1260, Chicago, Illinois 60611, USA. 2Center for Genetic Medicine, Northwestern University, 303 East Superior Room 7-125, Chicago, Illinois 60611, USA. Correspondence should be addressed to R.L.C. (r-chisholm@northwestern.edu). Published online 24 May 2007; doi:10.1038/nprot.2007.180

2007 Nature Publishing Group http://www.nature.com/natureprotocols

In this protocol, we present a standard method for extracting DNA from cells of the social amoeba Dictyostelium discoideum. While this procedure is similar to other phenol:chloroform-based purication methods, it is modied to account for the high level of carbohydrate and nucleases found in Dictyostelium cells. Genomic DNA can be isolated from wild-type and genetically modied cells using the described protocol, allowing molecular genetic analyses to be performed. Following cell lysis, nucleic acid extraction, and precipitation, the isolated DNA is suitable for digestion by restriction enzymes, amplication by PCR and Southern blotting. This procedure takes approximately 3 h to complete.

INTRODUCTION The social amoeba D. discoideum is a model research organism that is amenable to studying mechanisms of development1, chemotaxis2 and cell signaling3. The complete sequence of the Dictyostelium genome has been published4 and is held at dictyBase, a model organism database in which genomic data can be stored and annotated5. These newly available tools greatly facilitate the investigation of these biological processes through molecular genetic means. It is relatively simple to extract macromolecules (see Box 1) from D. discoideum, as the cells are protected only by a typical plasma membrane. Cells contain a large quantity of degradative enzymes6, and thus it is recommended to work on ice and as rapidly as possible to minimize the degradation of the samples. Dictyostelium DNA (see Box 1) can easily be isolated from cells; however, because of the high level of carbohydrates7,8, commercially available DNA preparation kits are not optimal. Most kits have been reported to result in very low yields (http://dictybase.org/ListServ_archive/ listserv_archive_molecbiol.html#genomic4). A very quick extraction of genomic DNA is possible; for this technique, see Charette and Cosson9 and Adley et al.10. This method is faster than the one described below; however, its applications are limited exclusively to

PCR analysis. To obtain a larger quantity of genomic DNA, a cesium chloride preparation11 is the preferred method, although the time and materials required for this protocol are much more extensive than that of our DNA preparation. Regardless of the method used, the absolute mass of the genomic DNA per cell is fairly small because Dictyostelium has a small genome size compared to higher eukaryotes (33.8 Mbp) (ref. 4). In the protocol presented here, we describe the extraction of DNA from Dictyostelium for the purpose of generating molecular constructs, which may be used in genetic manipulations, and for analysis of the resulting mutants. This method has been used successfully in our laboratory to generate and analyze mutant cell lines12,13. This relatively quick extraction protocol efciently produces genomic DNA that is suitable for all downstream applications, including PCR14, digestion with restriction enzymes and subsequent cloning11, and for Southern blot15 analysis. Considering the short time it takes to complete the following procedure and the basic materials required, the resulting DNA is of excellent quality. For molecular cloning applications, we have also provided protocols for DNA transformation16, along with techniques for culturing Dictyostelium cells17.

BOX 1 | FURTHER INFORMATION ABOUT DICTYOSTELIUM

1 108 Dictyostelium cells contain approximately18  22 mg nuclear genomic DNA  300 mg total RNA  11 mg total cellular protein  1.5 mg total carbohydrate Facts about Dictyostelium DNA4:  33.8 megabase pairs of nuclear genomic DNA in 6 chromosomes  AT content is 77.6%; GC content is 22.4%  Number of mRNA coding genes is approximately 12,500  Average gene size is 1,756 base pairs  69% of genes contain at least one intron  Average intron size is 146 base pairs  55 kilobase pairs of mitochondrial DNA19 (not included in this nuclear DNA preparation)  88 kilobase pairs of ribosomal DNA, which exists in many copies and represents up to 20% of the DNA in a cell20 (included in this nuclear DNA preparation)

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PROTOCOL
MATERIALS

. D. discoideum (http://dictybase.org/StockCenter/StockCenter.html) . Nuclei buffer: 20 mM Tris-HCl pH 7.4, 5 mM MgOAc, 0.5 mM EDTA

pH 8.0, 5% (w/v) sucrose . 20% (v/v) Triton X-100 in water . Proteinase K buffer: 100 mM Tris-HCl pH 7.4, 5 mM EDTA pH 8.0, 0.1 mg ml1 proteinase K, 1% (v/v) SDS m CRITICAL Add proteinase K just before each use. Shake gently or stir; mixing too vigorously will produce foam. . Phenol pH 8.0 (see REAGENT SETUP) . Chloroform ! CAUTION Chloroform is believed to be a carcinogen; avoid inhalation and use a fume hood while working with this reagent. . Phenol:chloroform (1:1) pH 8.0 (see REAGENT SETUP) . 100% (v/v) ethanol ! CAUTION Ethanol is extremely ammable. . TE buffer pH 7.4: 10 mM Tris-HCl pH 7.4, 1 mM EDTA . 10 mg ml1 RNase A (Sigma-Aldrich, cat. no. R6513) . 3 M NaOAc . 70% (v/v) ethanol EQUIPMENT . Centrifuge . Microfuge . 65 1C water bath

REAGENTS

. Vortex . Spectrophotometer
REAGENT SETUP Phenol pH 8.0 To 35 ml liqueed phenol, add 10 ml of 50 mM Tris pH 8.0. Shake vigorously and centrifuge at 2,500g for 10 min at room temperature (22 1C). Remove the upper layer (Tris), and repeat by adding 10 ml Tris, shaking, centrifuging and removing the Tris layer. Then cover the phenol with 5 ml Tris pH 8.0 and store at 4 1C in the dark (cover with aluminum foil if an opaque container is not available). It should be noted that ready-to-use phenol pH 8.0 may be purchased (Ambion; cat. no. AM9710). m CRITICAL The pH of phenol is very important; an acidic pH degrades DNA and thus partitions it to the organic phase. If the pH is below 7.0, the DNA may be lost owing to improper phase separation. Also, phenol oxidizes by a free radical process, indicated by a pinkish color. Do not use oxidized phenol, as it can result in DNA damage. ! CAUTION Phenol is caustic; always wear gloves and handle with extreme care. Phenol:chloroform (1:1) pH 8.0 Mix an equal volume of phenol pH 8.0 with chloroform. Store phenol:chloroform at 4 1C in the dark (cover with aluminum foil if an opaque container is not available). Premixed phenol:chloroform pH 8.0 may be purchased (Ambion, cat. no. AM9730). ! CAUTION Phenol:chloroform is caustic; always wear gloves and handle carefully. It is recommended to work in a fume hood while using phenol:chloroform.

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PROCEDURE 1| Grow and quantify Dictyostelium cells according to previously described methods17. m CRITICAL STEP It is recommended to use vegetative cells or cells developed in suspension for a short period to avoid cell aggregation, which makes lysis less effective and reduces the yield. Development for up to 6 h reduces the amount of cellular protein, giving a cleaner, larger yield of DNA. 2| Collect 23 107 Dictyostelium cells in a 50 ml polypropylene tube by centrifugation for 4 min at 500g at room temperature. Pour off the supernatant. Keeping the tube inverted, carefully drain the remaining supernatant onto a paper tissue. 3| Resuspend cells by gentle pipetting in 1 ml of nuclei buffer. Before adding the buffer, pellet may be loosened by scraping the tube over an uneven surface such as a microfuge tube rack. Transfer to a clean 1.5 ml microfuge tube. 4| Add 200 ml of 20% Triton X-100 and incubate on ice for 5 min to lyse cells. 5| Centrifuge at 12,000g (highest setting for a typical benchtop microfuge) for 5 min. Carefully remove the supernatant by pipetting. The pellet contains the nuclei. 6| Loosen pellet by brief vortexing and resuspend in 300 ml proteinase K buffer. 7| Incubate at 65 1C for 30 min. 8| Extract nucleic acids by adding an equal volume (300 ml) of phenol:chloroform (1:1). Invert the tubes to mix or gently vortex. m CRITICAL STEP Be careful not to vortex vigorously; this can lead to shearing of genomic DNA. 9| Centrifuge for 10 min at 12,000g. Following centrifugation, two distinct phases should be seen: the DNA will be contained in the aqueous (upper) layer, while the phenol:chloroform makes up the organic (bottom) layer containing mostly proteins. A distinct interphase composed of degraded cellular material will appear between the layers. 10| Carefully pipette the aqueous (upper) layer into a fresh 1.5 ml tube. m CRITICAL STEP Do not disturb the interphase while removing the aqueous layer. Failure to do so will result in a DNA sample containing proteins and other contaminants, which can have an inhibitory effect on subsequent enzymatic reactions. ! CAUTION Discard the tube containing the used phenol:chloroform according to your institutions hazardous waste guidelines. ? TROUBLESHOOTING 11| Extract the aqueous layer by adding one volume (300 ml) of chloroform. Invert the tubes or very gently vortex. This nal extraction with chloroform is intended to remove any remaining phenol as it is inhibitory for downstream applications. 12| Centrifuge for 5 min at 12,000g and carefully pipette the aqueous layer into a fresh 1.5 ml tube. 13| Precipitate the aqueous layer with 2.5 volumes (750 ml) of ice-cold 100% ethanol and let it stand at room temperature for 5 min or at 20 1C for longer. Salt does not need to be added to this precipitation step; the residual MgOAc from the nuclei buffer is sufcient. The subsequent precipitation (Step 16) does require the addition of salt, however.
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PROTOCOL
PAUSE POINT The genomic DNA sample will be stable at 20 1C for several months if you should choose to wait to complete the protocol. 14| Pellet DNA by centrifuging at 12,000g for 15 min at 4 1C. Remove the supernatant with a pipette without disturbing the pellet. The DNA will appear as a tight, whitish pellet at the bottom of the tube. Be sure to look for it before discarding the ethanol. 15| Resuspend pellet in 100 ml TE pH 7.4 containing 10 mg ml1 RNase A and incubate for 15 min at room temperature. 16| Precipitate again by adding 1/10 volume (10 ml) of 3 M NaOAc and 2.5 volumes (250 ml) of ice-cold 100% ethanol. Place the tube on ice for 15 min or longer at 20 1C. PAUSE POINT The DNA sample may also be stored at 20 1C at this point for a long period if necessary. 17| Centrifuge at 4 1C for 15 min at 12,000g and then remove the supernatant with a pipette without disturbing the pellet.
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18| Wash pellet with two volumes (200 ml) of ice-cold 70% ethanol and centrifuge for 2 min at 12,000g. 19| Carefully remove the supernatant with a pipette and leave the pellet to dry at room temperature. m CRITICAL STEP The pellet should not be allowed to dry too long. An over-dry pellet will be difcult to dissolve in TE. 20| Resuspend DNA pellet in 50 ml of TE pH 7.4. PAUSE POINT Store genomic DNA at 4 1C; DNA will remain stable for at least 3 months. m CRITICAL STEP Do not freeze, as repeated freezing and thawing in an aqueous solution will degrade genomic DNA. 21| Determine the DNA concentration by measuring the optical density (OD). Dilute DNA 1:5 in TE pH 7.4 and determine the OD260 using a spectrophotometer. Calculate the DNA concentration using the following formula: OD260 reading dilution factor ((50 mg ml1)/OD260 unit) DNA concentration (mg ml1). To calculate the DNA concentration in mg ml1, divide by 1,000. To determine the quality of the resulting DNA, take an OD280 reading, which is the optimum absorption for proteins. Pure DNA has an OD260/OD280 ratio in the range of 1.82.0. OD260/OD280 ratios below 1.8 indicate contamination with proteins and/or phenol. ? TROUBLESHOOTING TIMING This protocol takes approximately 3 h to complete. However, if more convenient, the DNA may be stored in ethanol at 20 1C at Step 13 and/or 16. ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1.
TABLE 1 | Troubleshooting table. Step 10 Problem Resulting DNA sample will not digest with restriction enzymes or yield PCR products Solution Some contaminants may have been transferred from the interphase when the aqueous layer was removed. Residual proteins can inhibit restriction enzymes and polymerases. Be conservative when pipetting off the aqueous layer, leaving a small amount (1020%) near the interphase Phenol might be old and oxidized. Check phenol:chloroform for color (should be clear, not pink; see REAGENT SETUP) and date; consider preparing a fresh solution if you feel its quality may be compromised This may be remedied through a re-extraction of the organic layer. To do this, add 100 ml of TE to the organic phase of Step 9, gently vortex and centrifuge for 10 min at 12,000g. Remove the aqueous layer and combine with the aqueous layer from the rst extraction. Then proceed to Step 11, making sure that volumes are adjusted in subsequent extractions and precipitations, through Step 14 OD readings greater than 1.0 are unreliable. Dilute the genomic DNA sample 1:10 rather than 1:5. Be sure to change the dilution factor to 10 in your calculation of DNA concentration OD readings less than 0.1 are unreliable. Dilute the genomic DNA sample 1:2 rather than 1:5. Be sure to change the dilution factor to 2 in your calculation of DNA concentration
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10

Resulting DNA sample will not digest with restriction enzymes or yield PCR products

10

Low DNA yield

21

OD260 reading is greater than 1.0

21

OD260 reading is less than 0.1

PROTOCOL
ANTICIPATED RESULTS Using 23 107 Dictyostelium cells, this protocol should yield approximately 35 mg of genomic DNA, or 3050 ng ml1 if resuspended in 50 ml of TE. The extracted DNA should be of fairly high quality, with an OD260/OD280 ratio of 1.8 or slightly above. See Figure 1 for typical results from this procedure. Notice that the uncut genomic DNA runs on the gel as a single band of high molecular weight; this is an indication of a good preparation. Degraded DNA will run as a smear on an agarose gel. Conversely, smearing of DNA cut with restriction enzymes indicates that the DNA is pure and can be well digested. A sample that digests poorly is of low quality and should not be used in further experiments.
Figure 1 | Results from the DNA extraction protocol. Genomic DNA (uncut, lane 1) was isolated from D. discoideum and digested overnight with EcoRI (lane 2) and HindIII (lane 3). A 100 ng portion of DNA was run in each lane on a 0.8% agarose gel. Banding occurs (lanes 2 and 3) owing to the large number of repetitive sequences present in the genome.
U nc ut Ec gen o R om I ic H D in N dI A II

10 kb 5 kb 2 kb 1 kb

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ACKNOWLEDGMENTS dictyBase (http://www.dictybase.org) is supported by grants from the NIH (GM64426 and HG00022). COMPETING INTERESTS STATEMENT The authors declare no competing nancial interests. Published online at http://www.natureprotocols.com Rights and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Chisholm, R.L. & Firtel, R.A. Insights into morphogenesis from a simple developmental system. Nat. Rev. Mol. Cell Biol. 5, 531541 (2004). 2. Firtel, R.A. & Chung, C.Y. The molecular genetics of chemotaxis: sensing and responding to chemoattractant gradients. BioEssays 22, 603615 (2000). 3. Mahadeo, D.C. & Parent, C.A. Signal relay during the life cycle of Dictyostelium. Curr. Top. Dev. Biol. 73, 115140 (2006). 4. Eichinger, L. et al. The genome of the social amoeba Dictyostelium discoideum. Nature 435, 4357 (2005). 5. Chisholm, R.L. et al. dictyBase, the model organism database for Dictyostelium discoideum. Nucleic Acids Res. 34, D423D427 (2006). 6. Cardelli, J.A., Golumbeski, G.S., Woychik, N.A., Ebert, D.L., Mierendorf, R.C. & Dimond, R.L. Dening the intracellular localization pathways followed by lysosomal enzymes in Dictyostelium discoideum. in Methods in Cell Biology. Volume 28: Dictyostelium discoideum: Molecular Approaches to Cell Biology (ed. J.A. Spudich) 139155 (Academic Press Inc., Orlando, FL, 1987). 7. West, C.M., van der Wel, H., Coutinho, P.M. & Henrissat, B. Glycosyltransferase genomics in Dictyostelium discoideum. in Dictyostelium Genomics (eds. W.F. Loomis & A. Kuspa) 2340 (Horizon Bioscience, Norfolk, 2005). 8. Nellen, W. et al. Molecular biology in Dictyostelium: tools and applications. in Methods in Cell Biology. Volume 28: Dictyostelium discoideum: Molecular Approaches to Cell Biology (ed. J.A. Spudich) 67100 (Academic Press Inc., Orlando, FL, 1987).

9. Charette, S.J. & Cosson, P. Preparation of genomic DNA from Dictyostelium discoideum for PCR analysis. BioTechniques 36, 574575 (2004). 10. Adley, K.E., Keim, M. & Williams, R.S.B. Pharmacogenetics: dening the genetic basis of drug action and inositol triphosphate analysis. in Methods in Molecular Biology. Volume 346: Dictyostelium discoideum Protocols (eds. Eichinger, L. & Rivero, F.) 517534 (Humana Press, Totowa, NJ, 2006). 11. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Woodbury, NY, 1989). 12. Fey, P., Stephens, S., Titus, M.A. & Chisholm, R.L. SadA, a novel adhesion receptor in Dictyostelium. J. Cell Biol. 159, 11091119 (2002). 13. Chen, P., Ostrow, B.D., Tafuri, S.R. & Chisholm, R.L. Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development. J. Cell Biol. 127, 19331944 (1994). 14. Mullis, K.B. & Faloona, F.A. Specic synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enymol. 155, 335350 (1987). 15. Southern, E.M. Detection of specic sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503517 (1975). 16. Gaudet, P., Pilcher, K.E., Fey, P. & Chisholm, R.L. Transformation of Dictyostelium discoideum with plasmid DNA. Nat. Protoc. 2, 13171324 (2007). 17. Fey, P., Kowal, A.S., Gaudet, P., Pilcher, K.E. & Chisholm, R.L. Protocols for growth and development of Dictyostelium discoideum. Nat. Protoc. 2, 13071316 (2007). 18. Ashworth, J.M. & Watts, D.J. Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture. Biochem. J. 119, 175182 (1970). 19. Ogawa, S. et al. The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content and genome organization. Mol. Gen. Genet. 263, 514519 (2000). 20. Sucgang, R. et al. Sequence and structure of the extrachromosomal palindrome encoding the ribosomal RNA genes in Dictyostelium. Nucleic Acids Res. 31, 23612368 (2003).

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