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 circle in
Saccharomyces cerevisiae). The enormous potential
and promising applications of plasmid compelled the

*Correspondent author
Telephone: 0184-2259503
Fax: 0184 2250042
E-mail: sachinandan@gmail.com

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 l of solution A
[Tris (50 mM; pH 8.0), EDTA (10 mM; pH 8.0) and
10mg/ml RNAse] and 300 l 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 l 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 l 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 l 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 M of each
primers, 1x PCR buffer, 1.5 mM MgCl2, 200 M of
dNTPs and 1 unit of Taq polymerase) and reaction
conditions {Initial denaturation 94C for 4 min,
94C for 30 s, annealing 52C for 30 s, extension
72C for 30 s, the reaction was run for 35 cycles
(from 2nd step to 4th step) and final extension of 72C
for 4 min} were used.

559

DNA yield and qualityThe 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 g,
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 PCRThe
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. 1Extracted 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. 2PCR 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-) from
plasmids isolated by using LH- 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,

ease in preparing and finally its safe to be kept

at room temperature as its working activity
remains intact even if kept more than a months 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.
References
1
2
3
4
5

6
7
8

Joshua Lederberg, Cell genetics and hereditary symbiosis,

Physiol Rev, 32 (1952) 403.
Birnboim H C & Doly J, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids
Res, 7 (1979) 1513.
Birnboim H C, A rapid alkaline extraction method for
the isolation of plasmid DNA, Methods Enzymol, 100 (1983)
243.
Holmes D S & Quigley M, A rapid boiling method for
the preparation of bacterial plasmids, Anal Biochem, 114
(1981) 193.
Grinsted J & Bennett P M, Preparation and electrophoresis
of plasmid DNA, in Methods in microbiology. Edited by J
Grinsted and P M Bennell Vol 21, (Academic Press London)
1988, 129.
Demeke T & Adams R P, The effect of plant polysaccharides
and buffer additives of PCR, BioTechniques, 12 (1992) 332.
Fang G, Hammar S & Grumet R, A quick and inexpensive
method for removing polysaccharides from plant genomic
DNA, BioTechniques,13 (1992) 52.
Pandy R N, Adams R P & Flourney L E, Inhibition of
random amplified polymorphic DNAs (RAPDs) by plant
polysaccharides, Plant Mol Bio Reporter, 14 (1996) 17.