A novel method of plasmid isolation using laundry detergent

Indian Journal of Experimental Biology Vol. 49, July 2011, pp. 558-560 A novel method of plasmid isolation using laundry detergent P Yadav1, A Yadav2, V Garg3, T K Datta1, S L Goswami1, & S De1*, 1 Animal Biotechnology Centre and 2Animal Biochemistry Division, National Dairy Research Institute, Karnal 132 001, India. 3 Department of Biosciences and Biotechnology, Banasthali University, Banasthali 304 022, India. Received 1 April 2009; revised 1 February 2011 Since the discovery of plasmid, various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction at pH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl sulfate is replaced by lipase enzyme present in laundry detergent. A good plasmid preparation can be made, which is well suited for subsequent molecular biology applications. By taking safety measures on count, contaminants like, RNA and protein can be completely avoided with maximized plasmid yield. The resultant plasmid quality and quantity can be well comparable to other prevalent methods. Keywords: Plasmid, Laundry detergent, Vector In 1952 Joshua Lederberg, while reviewing the literature on cell heredity, suggested the term “Plasmid" first time as an extra chromosomal hereditary determinants1. Since then throughout the world, researchers found the answer of the presence of plasmid in bacterial cells. Plasmids are circular double-stranded extra chromosomal cytoplasmic molecules that replicate independently of the bacterial chromosome as they are not essential for the bacterium but may confer a selective advantage to the bacterium harboring them, such as the ability to make the bacterium antibiotic resistant. Naturally plasmid occurs in bacteria and is sometimes found in eukaryotic organisms (e.g., the 2 µ circle in Saccharomyces cerevisiae). The enormous potential and promising applications of plasmid compelled the —————— *Correspondent author Telephone: 0184-2259503 Fax: 0184 2250042 E-mail: [email protected] researchers to make use of plasmid in molecular biology. With the advent of restriction enzymes in 1970s, utility of plasmid in molecular biology revolutionized and techniques like recombinant DNA technology or genetic engineering came into existence. The use of high quality plasmid DNAs often determines the success in various manipulations of genetic material during routine applications such as polymerase chain reaction (PCR) amplification, DNA sequencing and sub-cloning of transgenes. For all the above mentioned techniques isolation of highly purified plasmid DNA from E. coli is an essential step. Traditional methods of plasmid isolation are the alkaline lysis2, 3 and the boiling4 methods. Extraction process is based on the size, conformation and density of plasmids. All the extraction processes vary in subtle ways; they all have three main stages that is lysis of bacterial cells, selective release of plasmid DNA from cell matrices, and removal of such contaminants to recover plasmid DNA5. In general practice, problems are often linked to the isolation of pure (high quality) plasmid DNA. These problems often arise due to contamination by phenolic compounds and polysaccharides as they inhibit classical primer PCR by inhibiting Taq DNA polymerase activity6-8. These contaminations distort the results in many analytical applications and therefore lead to wrong interpretations. Priorities of every developed protocol are high yield and good quality of plasmid involving less time and expenditure. In the present study a protocol using detergent has been developed. In this detergent method cells are lysed by lipase enzymes, chromosomal DNA is denatured by the strong alkali (NaOH), and the proteases present in detergent will additionally help phenol for digestion and removal of proteins. Presence of protease in detergent is an added advantage in isolating protein free plasmid DNA. The plasmid samples used in the present experiment were recombinant pTarget vector (mammalian vector) having insert of luteinizing hormone-alpha gene of buffalo (Bubalus bubalis). The desired plasmid was propagated in a suitable competitive host cell (E. coli strain XL-1 Blue). For isolation of plasmid, 10 ml of LB medium was inoculated with recombinant E. coli culture and allowed to grow (overnight-14 to 16 hr). NOTES Pellet was obtained by centrifuging 3 ml overnight culture of E. coli strain XL-1 Blue at 10,000 g for 2 min. The pellet was dissolved in 300 µl of solution A [Tris (50 mM; pH 8.0), EDTA (10 mM; pH 8.0) and 10mg/ml RNAse] and 300 µl of solution B (0.2% of laundry detergent-Surf Excel and 10 N NaOH) and allowed to stand at room temperature for 3-5 min. After the incubation, 3M potassium acetate (pH 5.2) was added and again incubated on the ice for 15-30 min. Following incubation, this solution was centrifuged at 10,000 g, for 15 min, and the supernatant was collected in a fresh microfuge tube. To the supernatant 500 µl of phenol (pH 8.0) and chloroform solution (1:1) was added and was allowed to remain at room temperature for 4-5 min. Thereafter microfuge tube was centrifuged for 10 min at 10,000 g, and aqueous phase was collected. The plasmid DNA was precipitated by adding equal volume of isopropanol to the supernatant and centrifuged at 10,000 g for 15 min. The obtained pellet was washed with 70% ethanol, and dissolved in 20 µl of nuclease free water. Altogether 50 positive clones were selected having pTarget vector with luteinizing hormone alpha gene for validation of this plasmid isolation protocol using the above mentioned procedure. Isolated DNA samples were analyzed by Nanodrop spectrophotometer (Bio-Tek instruments, Inc, SaintQuentin en Yvelines, France). DNA concentration was determined by taking absorbance at 260 nm (A260). The purity of the DNA was determined from the ratio of A260/A280. The quality of the isolated plasmid DNA was also evaluated by running 3 µl of isolated DNA on 1% agarose gel. The intactness of plasmid DNA was confirmed by PCR amplification of luteinizing homone-alpha subunit using gene specific primers of Luteinizing hormone-alpha subunit (321 bp). Primers for luteinizing hormone-alpha were designed using web based software PRIMER-3 (http://wwwgenome.wi.mit.edu/cgi- bin/prime/primer3-ww.cgi) and custom synthesized from M/s Sigma-Aldrich, USA. Optimized PCR chemicals (0.2 µM of each primers, 1x PCR buffer, 1.5 mM MgCl2, 200 µM of dNTPs and 1 unit of Taq polymerase) and reaction conditions {Initial denaturation 94°C for 4 min, 94°C for 30 s, annealing 52°C for 30 s, extension 72°C for 30 s, the reaction was run for 35 cycles (from 2nd step to 4th step) and final extension of 72°C for 4 min} were used. 559 DNA yield and quality—The purity of isolated plasmid was measured by spectrophotometer and confirmed on gel (1% agarose) electrophoresis. The purity ratio of plasmid was 1.7 at absorbance 260/280 nm (A260/A280) and total yield was 20 µg, which clearly demonstrated the high quality and quantity of isolated DNA. Sharp intact band of all three forms of plasmid on agarose gel showed the good integrity of genomic DNA (Fig. 1). Integrity of DNA confirmed by PCR—The applicability of the present method for downstream applications was checked by amplifying the LH alpha gene from the isolated recombinant plasmid DNA by PCR. In all the samples tested, a single band of the amplicon of ~321 base pairs was observed (Fig. 2). Fig. 1—Extracted plasmid DNA (pTarget with luteinizing hormone- alpha gene) from five different bacterial clones using the simplified protocol using laundry detergent. L1-L5 represents the positive clones of plasmid (pTarget with luteinizing hormonealpha gene), isolated with the laundry detergent. Fig. 2—PCR amplification of luteinizing hormone-alpha gene insert using extracted plasmid DNA as a template. Lane M represents the 100 bp marker and lane no. 1-6 represents the amplified luteinizing hormone-alpha subunit (LH-α) from plasmids isolated by using LH-α gene specific primers. 560 INDIAN J EXP BIOL, JUlY 2011 In the present study presence of enzymes like lipases and proteases in laundry detergent have been exploited to isolate plasmid DNA of high yield and quality. Enzyme lipase efficiently ruptures the cell membrane and thus replaces the use of sodium dodecyl sulphate (SDS) required for cell lysis in other available method; on the contrary proteases present in laundry detergent increases the efficiency of phenol and chloroform to separate cellular proteins. These enzymes synergistically function with phenol in digestion and removal of proteins, thus ensure isolated plasmid DNA of high quality. Both low and high copy number plasmids isolated with this method are largely super coiled and had the optical density (OD) ratio of 1.7-2.0 at 260/280 nm. The integrity check of the plasmid was done by slab gel electrophoresis. To ensure the quality check of the plasmid, PCR was performed using tested luteinizing hormone alpha gene specific primers. The amplified products analyzed on agarose gel. The presence of single band of the expected size ensures usability of isolated plasmid DNA for downstream applications. Along with its efficiency of extracting high quality plasmid DNA, the inclusion of laundry detergent in plasmid extraction protocol has certain advantages viz. solution is very cost effective, easily available, View publication stats ease in preparing and finally it’s safe to be kept at room temperature as its working activity remains intact even if kept more than a month’s time at room temperature. This method is largely useful for the researchers who have scarcity of SDS or want to escape preparing its solution as its preparation is tedious. 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