AsPac
Biol.J.Biotechnol.
164 J. Mol.
AsPac
Mol. Biol.2014
Biotechnol. Vol. 22 (1), 2014
Vol. 22 (1) : 164-170
Simple DNA Isolation Protocols for Cassava
Sample preservation procedures and simple DNA isolation protocols
for the tuberous crop, cassava (Manihot esculenta Crantz.)
Nuri Kiptantiyawati1, Nawar Lina Syarifah1, Pradita Maulia2, Etik Sulistiyowati1,
Mirza Ramadhana Putra1, Yudiansyah1, Nurul Khumaida1, Sintho Wahyuning Ardie1*
Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University (IPB),
Jl. Meranti Kampus IPB Darmaga 16680, Bogor, West Java, Indonesia.
2
Department of Biochemistry, Faculty of Mathematics and Natural Science, Bogor Agricultural University (IPB),
Jl. Agatis Kampus IPB Darmaga 16680, Bogor, West Java, Indonesia.
1
Received 8th September 2013/ Accepted 10th February 2014
Abstract. Large scale collection of samples from remote areas followed by efficient DNA isolation is often required for genetic
studies of a crop species using molecular methods. In this paper, we describe the comparison of different tissue preservation
procedures, and develop a reliable and efficient protocol for isolating genomic DNA from four cassava (Manihot esculenta
Crantz.) genotypes. We found that cassava leaf tissue can be preserved at 4oC for one week without significant loss in yield
or quality. Comparison of six DNA isolation protocols revealed that a simplified version of an existing protocol using CTAB
buffer was the best method to isolate DNA from leaf samples of ‘Ratim’, ‘Jame-jame’, ‘Malang-4’ and ‘Gajah’ genotypes. The
methodology not only obtained high DNA yields of 771, 585, 542 and 2,672 mg DNA/g of sample, respectively, but also
produced high quality DNA with A260/A280 ratios of 1.9, 1.9, 1.7 and 2.0, respectively. PCR amplification using the
resulting DNA produced reliable and reproducible results, indicating the suitability of DNA for subsequent PCR analyses. This
simplified protocol using CTAB buffer removes the necessity for grinding using liquid nitrogen and the use of phenol, PVP
(polyvinyl polypyrrolidone), and b-mercaptoethanol in the isolation step, and can thus be considered technically easy and
cost-effective.
Keywords: Cassava, DNA extraction, DNA quality, DNA quantity, Sample handling, Tuber crop.
INTRODUCTION
Cassava (Manihot esculenta Crantz., Euphorbiaceae)
is an important dietary carbohydrate source for
approximately 800 million people in the tropics (Liu et al., 2011).
Africa is the major producer of cassava worldwide,
followed by Asia and Latin America, with total production
being around 200 million tonnes (FAO, 2010). Although
the storage root of cassava is rich in starch (74-85% by
dry weight) (Taylor et al., 2004), it is very poor in protein
(Nassar et al., 2009) and has variable levels of linamarin
content and cyanogenic potential (Yeoh and Truong,
1993). In Africa, cassava production is often hampered by
cassava mosaic disease (CMD), a major viral disease of
cassava that can be easily transmitted via infected stem
material during vegetative propagation (Legg and Fauquet,
2004). Improvement of both yield and nutritional quality,
reduction in cyanide content, increased virus resistance,
and delayed deterioration of the storage roots are among
the agronomic characters targeted in cassava breeding
programmes.
Breeding of clonally propagated non-inbred crops, such
as cassava, can be made more efficient through the use of
DNA markers. DNA markers are powerful tools in crop
improvement programmes for various important crops
(Collard et al., 2005) including cassava (Fregene et al.,
2001). Several molecular markers have been developed in
cassava, including RAPD markers related to cyanogenic
potential (Wong et al., 1997), microsatellite markers
to detect cassava variability (Chavarriaga-Aguirre et al.,
1998; Montero-Rojas et al., 2011), and SSR markers
related to CMD resistance (Bi et al., 2010). Isolation of a
sufficient quality of high quality DNA is a prerequisite for
successful DNA marker implementation for breeding
strategies. However, good quality DNA isolation from
leaves of tropical tuber crops is often complicated due to the
presence of phenolic compounds and polysaccharides
(Sharma et al., 2008). Oxidized phenolics will irrevers* Author for correspondence: Sintho Wahyuning Ardie, Department of
Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural
University (IPB), Jl. Meranti, Kampus IPB Darmaga, Bogor, West Java,
Indonesia 16680.
Tel. and Fax: +62-251-8629353, E-mail - [email protected].
AsPac J. Mol. Biol. Biotechnol. Vol. 22 (1), 2014
ibly bind to protein and nucleic acids during tissue
homogenisation (Loomis, 1974), thus decreasing DNA
availability for subsequent analyses such as restriction
enzyme digestion and PCR (Porebski et al., 1997).
Inhibition by polysaccharides of restriction enzyme
digestion and PCR amplification has also been reported
(Do and Adams, 1991; Fang et al., 1992). In addition,
widespread application of molecular marker techniques
often necessitate the isolation of DNA from a large number
of samples and sometimes requires collection of samples
from remote locations. There have only been limited studies
regarding efficient DNA isolation protocols developed for
cassava (Sharma et al., 2008; Bhattacharjee et al., 2010).
These studies involve elaborate and time consuming steps
such as long incubation at 65oC or 37oC, use expensive
chemicals such as proteinase K or liquid nitrogen, or involve
the use of specific equipment designed specifically for DNA
isolation. Moreover, these studies were conducted only on one
cassava genotype. Therefore, identification of simple sample
collection techniques and an efficient DNA isolation
procedure for cassava is of high importance. Here we
present a comparison between different sample preservation
procedures and modified DNA isolation protocols for four
cassava genotypes.
MATERIALS AND METHODS
Plant Materials Collection and Preservation.
Four
cassava genotypes (one Indonesian national variety
‘Malang-4’; two local genotypes from North Halmahera
Regency, Indonesia: ‘Ratim’ and ‘Jame-jame’; and one
local genotype from East Kalimantan Province,
Indonesia: ‘Gajah’) were planted in the Cikabayan
Experimental Field, Bogor Agricultural University (240 m
asl). Young leaftissues (apical leaves or leaves at the three
uppermost nodes) of those cassava genotypes were
collected directly from the field, wrapped in aluminum
foil and kept in an ice box. The experiment was arranged in
completely randomised design with two groups of treatments
and three replicates. The first group of treatments investigated
sample preservation procedures: control (DNA isolated
directly from the freshly harvested leaf tissues), storage at
-20oC for one week prior to DNA isolation, and storage
at 4oC for one week prior to DNA isolation. The second
group of treatments investigated the DNA isolation
protocol: standard protocol using CTAB or SDS buffer,
simplified protocol using CTAB or SDS buffer, and
simplified protocol with additional heating using CTAB or
SDS buffer. Each sample was weighed prior to preservation
or DNA isolation.
Reagents for DNA Extraction. Two DNA extraction
buffers (CTAB-cetyltrimethylammonium bromide and
SDS-sodium dodecyl sulphate) were tested in this study.
Simple DNA Isolation Protocols for Cassava
165
The CTAB buffer consisted of 2% CTAB, 100 mM
Tris-HCl (pH 8.0), 20 mM EDTA (pH 8.0), and 1.4 M
NaCl (Doyle and Doyle, 1987), while the SDS buffer
consisted of 0.5% SDS, 200 mM Tris-HCl pH 8.0, 25 mM
EDTA, and 0.25 M NaCl (Edwards et al., 1991). The other
reagents required were CI (chloroform : isoamyl alcohol,
24:1, v/v), and ethanol (100% and 70%, stored at 4oC).
DNA Extraction Protocols.
Standard protocol. The standard protocol referred
to Doyle and Doyle (1987) was employed after slight
modification. Leaf samples ((± 0.2 g) were ground in 700
mL CTAB or SDS lysis buffer in a mortar. Homogenised
samples were transferred into 1.5 mL microtubes and were
incubated at 65oC in a water bath for 30 minutes. After 10 minutes incubation at room temperature an equal
volume of CI (24:1) were added and mixed by gentle
inversion. The samples were centrifuged at 10,000 rpm for
5 minutes at 4oC and the supernatant was transferred to a
fresh tube. DNA precipitation was done by adding 2 times
volume of cold 100% ethanol. The obtained DNA pellet
was air-dried until the ethanol was evaporated and then
dissolved in an appropriate amount of water (50-150 mL).
Simplified Protocol .
The simplified protocol was a
modification from the standard protocol, particularly
excluding the grinding step in a mortar and the incubation
step at 65oC. Leaf samples ((± 0.01g) were briefly ground
using a plastic mini pestle in 1.5 mL microtube containing
50 mL lysis buffer (CTAB or SDS). Additional lysis buffer
(350 mL) was added and the samples were incubated at room
temperature for 10 minutes followed by centrifugation at
10,000 rpm for 5 minutes at 4oC. Supernatant was transferred
to a fresh tube. DNA precipitation was done by adding 2 times
volume of cold 100% ethanol. The obtained DNA pellet was
air-dried until the ethanol evaporated and then dissolved in
an appropriate amount of water (50-100 mL).
DNA Quantification and Analysis. Absorbance at 260
nm (A260) was measured to determine DNA quantity and
calculation values of A260/280 and A260/230 ratio to
estimate DNA purity of each sample using NanoDrop
2000 UV-Vis spectrophotometer (Thermo Scientific,
Wilmington, DE). Data of DNA concentration (mg/g
FW) and quality (A260/280 and A260/230 ratio) obtained were
analyzed using ANOVA and significant means were
compared using Duncan’s multiple range test (p<0.05).
To check the suitability of extracted DNA for downstream
analysis, RAPD analysis was done with the E19 primer
(5’- GTGACCAGCC -3’). Each 20 mL of PCR reaction
mixture of consisted of 5 mL (60 ng/ 20 mL) genomic DNA,
10 mL PCR master mix, and 5 mL primer (50 pmol/ 20
mL). The PCR master mix (Kapa Biosystem, Massachusetts,
US) consisted of 50 mM KCl, 10 mM Tris HCl pH 9.1,
tritonTMx-100 0.01 %, 0.08 M dNTP, 1.5 mM MgCl2,
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Simple DNA Isolation Protocols for Cassava
and 2-3 unit DNA polymerase. The PCR cycle consisted of
initial denaturing at 94oC for 5 minutes, followed by 45
cycles at 94oC for 5 seconds, 35oC annealing for 30 seconds, and
extension at 72 oC for 30 seconds. The final extension step
was done at 72oC for 10 minutes, then the PCR products
were stored at 4oC. Amplified products were electrophoresed
on 1% agarose gels in a half volume of Tris-acetate-EDTA
(TAE) buffer, followed by ethidium bromide staining and
visualized under an UV transilluminator (Alpha Innotech
Corp., CA).
RESULTS AND DISCUSSION
Sample preservation procedures.
Different sample
preservation procedures did not significantly affect the
concentration, yield or quality (A260/280 and A260/230) of
DNA isolated from ‘Ratim’, ‘Jame-jame’, and ‘Gajah’
genotypes, however the yield and quality of DNA isolated
from ‘Malang-4’ genotype was affected by the sample
preservation procedure (Table 1). DNA isolated from
freshly harvested leaves of the ‘Malang-4’ genotype
had a higher yield compared to those isolated from
samples preserved for one week at 4oC or - 20oC.
Plant DNA isolation for downstream application such
as DNA digestion, amplification and cloning was often
complicated by the presence of secondary metabolites and
polysaccharides in the tissue. A good and suitable DNA
isolation method for any plant material should produce not
only high DNA yield but also good DNA quality.
The A260/280 ratio is generally used to indicate DNA
purity, especially from protein contamination, and a ratio
between 1.8-1.9 indicates good DNA quality (Sambrook
et al., 1989). DNA isolated from cassava leaves using CTAB
methods developed by Doyle and Doyle (1987) contains
high levels of protein contaminants as reported by Sharma
et al. (2008). In this study, the A260/280 ratio of DNA
isolated from the leaves of ‘Ratim’ and ‘Gajah’ genotypes,
freshly harvested or stored for one week at 4oC or -20oC,
ranged from 1.8 to 1.9 (Table 1). In contrast, the A260/280
ratio of DNA isolated from the leaves of ‘Jame-jame’ and
‘Malang-4’ genotypes were <1.8. Since the A260/280 ratio was not
affected by the sample preservation procedure (Table 1);
this result indicates that DNA purity from protein
contamination was more affected by genotype.
Bhattacharjee
et al. (2009) used a NaCl-CTAB
-azide solution to preserve cassava leaves for one week at
4oC prior to DNA isolation and stated that the quality of
DNA obtained was high. However, that study did not
report the A260/A280 ratio value. Our study clearly showed
Table 1. Concentration, yield and quality of DNA obtained from the leaves of four cassava genotypes using
different sample preservation procedures.
DNA concentration
(ng/ mL)
DNA yield
(mg/ g FW)
Ratio A260/A280
Ratio A260/A230
‘Ratim’ genotype
Fresh sample
Stored for one week at 4oC
Stored for one week at -20oC
819
827
1,420
1,317
1,339
1,822
1.88
1.82
1.93
1.23
1.06
1.23
‘Jame-jame’ genotype
Fresh sample
Stored for one week at 4oC
Stored for one week at -20oC
1,001
799
1,208
2,769
1,559
1,828
1.69
1.73
1.72
1.06
0.89
0.89
‘Malang-4’ genotype
Fresh sample
Stored for one week at 4oC
Stored for one week at -20oC
1,241
911
1,741
4,167a
2,430b
1,960b
1.58b
1.71a
1.60b
0.78b
1.03a
0.87b
‘Gajah’ genotype
Fresh sample
Stored for one week at 4oC
Stored for one week at -20oC
2,442
3,073
3,701
4,256
4,158
6,006
1.79
1.90
1.80
1.07
1.31
1.21
Sample preservation
Note : Values with different letters in each column and each genotype indicate significant difference by
Duncan’s Multiple Range Test, P < 0.05.
AsPac J. Mol. Biol. Biotechnol. Vol. 22 (1), 2014
that the use of a preservation solution (e.g. NaCl-CTAB
-azide solution) is not necessary for certain cassava
genotypes such as ‘Ratim’ and ‘Gajah’ which had an A260/A280
ratio of > 1.8. Polysaccharides are the other compound that
are often found in the tissue of tropical tuber crops (Sharma
et al., 2008). The A260/A230 ratio indicates the contamination
rate of contaminants other than proteins, especially
polysaccharides (Wilson and Walker, 2005). DNA
isolated from the four cassava genotypes, irrespective to the
preservation procedure, had an A260/A230 ratio of <1.7,
indicating the high contamination of polysaccharides.
Concentration, yield and quality of DNA were not
affected by the sample preservation procedure used in
this study, except from the ‘Malang’ genotype. Preserving
samples at 4oC could reduce the need for liquid nitrogen,
storage at -20°C, and the use of preservation solution
(e.g. NaCl-CTAB-azide solution), thus reducing the
cost involved in preservation of leaf samples over longer
periods. This procedure would be beneficial when samples are
collected from remote areas and direct sample processing is
difficult.
DNA isolation protocols.
DNA isolation protocol
significantly affected the concentration, yield and quality
(A260/A280 and A260/A230) of DNA isolated from the four
cassava genotypes (Table 2). The simplified protocol and
simplified protocol with additional heating using SDS
buffer yielded a higher amount of DNA compared to the
other protocols, but lower amounts of DNA compared to
other protocols in ‘Jame-jame’, ‘Ratim’, and ‘Malang-4’
genotypes. Sodium dodecyl sulfate (SDS) is a strong
anionic detergent that can solubilise proteins and
lipids (Tan et al., 2013). The CTAB and SDS buffer showed
similar efficiency when used in standard protocols where the
samples were finely ground in a mortar. Therefore, in the
simplified protocols where leaf samples were only roughly
cut into small pieces SDS might be more efficient in lysing
the protein component of the cell membrane than CTAB.
Our results showed that DNA isolation protocol did not
significantly affect the A260/A280 ratio of ‘Ratim’ genotype
(Table 2). High quality DNA (with a A260/A280 ratio >1.8)
was successfully obtained from the DNA of the ‘Ratim’
genotype using the six isolation protocols, while in other
genotypes high quality DNA (A260/A280 > 1.8) were found
mainly in DNA isolated using the standard protocol
using CTAB buffer.
DNA isolated from ‘Ratim’
and ‘Jame-jame’ genotypes using the standard CTAB
protocol produced an A260/A230 ratio > 1.7, however the
other protocols resulted in low A260/A230 ratios. The low
A260/A230 ratio measured in this study, especially in DNA
isolated using the simplified protocols, might be because
the samples were not finely ground. Sharma et al. (2008)
finely ground the cassava leaf tissue using liquid nitrogen,
while Bhattacharjee et al. (2009) finely ground the cassava
leaves in a GenoGrinder 2000. In the simplified protocols
developed in this study, leaf samples were only roughly cut
into small pieces to reduce the time-consuming grinding
Simple DNA Isolation Protocols for Cassava
167
step or the need for special grinding apparatus. Generally,
the use of a strong detergent such as SDS in the simplified
protocols yielded higher amount of DNA compared to the
protocol using CTAB. However, the use of SDS in the
simplified protocols produced DNA with lower
uality (judged by A260/A280 and A260/A230 ratios)
compared to those isolated with CTAB. Since DNA quality
is more important than DNA quantity in determining the
optimal DNA isolation protocol (Tung-Nguyen et al., 2009),
CTAB buffer is more recommended than SDS buffer for
use in the simplified protocols. Furthermore, an additional
heating step seems not to be necessary in the simplified
protocol using CTAB buffer since additional heating neither
resulted in higher quantity nor quality of DNA.
The DNA isolation protocols developed in this study
included some modifications to simplify the general
steps and reduce the overall cost of the experiment. Most
protocols for DNA isolation from cassava use phenol:
chloroform: isoamyl-alcohol (25:24:1) to increase DNA
purity (Doyle and Doyle, 1987; Sharma et al., 2008).
In this study we exclude the use of phenol because of
its caustic and toxic properties. Previous studies also
demonstrated successful DNA isolation from tissue
containing high levels of secondary metabolites and
polysaccharides (Bhattacharjee et al., 2009; Sahu et
al., 2012). We also exclude the use of several chemical
reagents in our protocols. The extraction buffers used in this
study did not contain PVP (polyvinyl polypyrrolidone) or
b-mercaptoethanol as used by Sharma et al. (2008) and
Bhattacharjee et al. (2009) or liquid nitrogen and proteinase
K as used by Sharma et al. (2008) for extracting genomic
DNA from cassava leaves yes still produced comparable
results to those two previous studies. Reduction of the
number of chemical reagents used in the DNA isolation
protocols would considerably reduce the overall cost of
experiments. Simplification in the DNA isolation steps was
also done in this study to reduce the time consuming steps in the
process. Sharma et al. (2008) used proteinase K in their DNA
isolation protocol and thus needed to perform incubation
steps (30 minutes at 37oC and 30 minutes at 65oC). In
this study we did not use PEG or proteinase K in the DNA
isolation protocols and successfully obtained high quality
DNA (A260/A280 ratio > 1.8). Finally, as we did not involve
the use of specific equipment such as the GenoGrinder
2000 used by Bhattacharjee et al. (2009), our methods can
be applied in laboratories where such sophisticated pieces of
equipment are unavailable.
PCR analysis.
Spectrophotometry-based DNA
quantification and quality analyses, such as Nanodrop,
can be affected by external factors and sometimes may
not reflect the actual quality and quantity of DNA
extracted. Therefore, the evaluation of a newly developed
DNA isolation protocol should be based on the success of
downstream analyses using the isolated DNA. PCR analyses
using the E19 primer were conducted in order to evaluate
the suitability of isolated DNA for downstream analyses such
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Simple DNA Isolation Protocols for Cassava
Table 2. Concentration, yield and quality of DNA from the leaves of four cassava genotypes by different
DNA isolation protocols.
DNA
concentration
(mg/ mL)
DNA yield
(mg/ g FW)
Ratio A260/A280
Ratio A260/A230
‘Ratim’ genotype
Standard CTAB
Standard SDS
Simplified CTAB
Simplified SDS
Simplified CTAB + heating
Simplified SDS+ heating
3,155a
2,475a
31b
215b
48b
210b
1,147b
1,211b
771c
2,748a
508c
2,573a
1.98
1.84
1.89
1.81
1.92
1.82
1.73a
1.56a
0.88c
1.17b
0.83c
0.88c
‘Jame-jame’ genotype
Standard CTAB
Standard SDS
Simplified CTAB
Simplified SDS
Simplified CTAB + heating
Simplified SDS+ heating
2,520a
2,271a
67c
614b
54c
488bc
1,348b
1,346b
585c
5,108a
289c
3,635a
2.00a
1.76bc
1.91ab
1.44d
1.72c
1.45d
1.93a
1.15b
0.94bc
0.44d
0.79c
0.42d
‘Malang-4’ genotype
Standard CTAB
Standard SDS
Simplified CTAB
Simplified SDS
Simplified CTAB + heating
Simplified SDS+ heating
1,877b
3,117a
114c
1,646b
113c
921bc
1,098bc
1,629b
542dc
6,004a
532d
7,308a
1.87a
1.91a
1.68b
1.35c
1.64b
1.35c
1.24a
1.27a
0.97b
0.46c
0.89b
0.53c
‘Gajah’ genotype
Standard CTAB
Standard SDS
Simplified CTAB
Simplified SDS
Simplified CTAB + heating
Simplified SDS+ heating
5,570a
7,819a
600b
2,584b
484b
1,376b
2,747b
3,696b
2,672b
8,218a
2,311b
9,200a
1.90ab
1.90ab
1.98a
1.68bc
1.88ab
1.62c
1.41ab
1.27b
1.62a
0.86c
1.39ab
0.66c
DNA isolation protocol
Note : Values with different letters in each column and each genotype indicate significant difference
by Duncan’s Multiple Range Test, P < 0.05.
as PCR. Since the sample preservation procedure used did
not significantly affect the DNA quality of the four cassava
genotypes (Table 1), PCR analyses were done only for
‘Jame-jame’ and ‘Gajah’ genotypes that were preserved
at -20oC for one week. To show the efficiency of DNA
isolation protocols in this study, DNA isolated using
various DNA isolation protocols from ‘Jame-jame’ and
‘Gajah’ genotypes that were preserved at -20oC were
used in the PCR analyses. Successful and reproducible
amplifications were obtained in the DNA samples isolated
from ‘Jame-jame’ and ‘Gajah’ genotypes using various DNA
isolation protocols (Figure 1). This result indicates that
despite the low A260/A230 ratio, the quantity obtained was
high and quality was adequate for downstream applications
such as RAPD analysis. However, with advances in genomics
and use of next gene sequencing methods, even the presence
of small quantities of impurities may prevent good results
be obtained by these sophisticated machines. Therefore, the
results obtained in this study need to be further evaluated
for other downstream analyses such as enzyme digestion and
sequencing analyses.
DNA yield and quality variability between
genotypes. Our results showed that DNA yield and quality
varied between cassava genotypes. The optimal sample
preservation procedure and DNA isolation protocol depends
on the cassava genotype. For example, storage at -20oC
for one week significantly reduced the DNA yield and
quality of ‘Malang-4’ genotype but not in other
genotypes (Table 1). Moreover, although the simplified CTAB
protocol resulted in the best DNA yield and quality isolated
from all cassava genotypes used in this study, the simplified
AsPac J. Mol. Biol. Biotechnol. Vol. 22 (1), 2014
Simple DNA Isolation Protocols for Cassava
169
Figure 1.RAPD assay. Conducted with the DNA isolated from ‘Jame-jame’
genotype (A) or ‘Gajah’ genotype (B) preserved at -20oC for one week.
Amplification was by primer E-19. M = 1 kb marker, 1 = standard CTAB,
2 = standard SDS, 3 = simplified CTAB, 4 = simplified SDS, 5 = simplified
CTAB with additional heating, 6 = simplified SDS with additional heating.
protocol and simplified protocol with additional heating
using CTAB buffer yielded lower amounts of DNA
compared to other protocols in three genotypes (namely,
‘Jame-jame’, ‘Ratim’, and ‘Malang-4’ genotypes) but not
in the ‘Gajah’ genotype (Table 2). Sharma et al. (2008)
reported variation in DNA yield and quality isolated from
different tropical tuber crop species. Our results further
indicate that the suitable sample preservation procedure
and DNA isolation protocol for cassava might be genotype
-specific.
The simplified DNA isolation protocol using CTAB
buffer described here is rapid, technically easy and not labour
intensive. The key modifications to the standard protocol
are 1) excluding the use of phenol, thus reducing the
hazardous nature of the procedure, 2) the simplified
DNA isolation protocol is carried out in Eppendorf tubes
to minimise the chances of contamination and loss of
DNA, 3) excluding the use of several expensive chemical
reagents (i.e. liquid nitrogen, PVP, b-mercaptoethanol, and
proteinase K) to reduce the overall cost of the experiment,
4) excluding the need for specific equipment, thus
it is suitable to be used in small laboratories where
sophisticated equipment is unavailable. The simplified
protocol using CTAB buffer can therefore be used for large
scale cassava DNA isolation, and cassava leaves can be
preserved at 4oC prior to DNA isolation especially when the
leaf tissues are collected from remote areas
ACKNOWLEDGEMENTS
This work was supported by Directorate General of Higher
Education, Ministry of Education and Culture, Indonesian
Republic through Student Creativity Program for Research
(PKM-P) 2013.
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