Reports

eDNA extraction protocol for metagenomic

studies in tropical soils
Fabián Echeverrı́a-Beirute*,1 , Ingrid Varela-Benavides1 , Jose P Jiménez-Madrigal1 , Milagro Carvajal-Chacon2 & Tomás
Guzmán-Hernández1
1
Instituto Tecnológico de Costa Rica, Campus Tecnológico Local San Carlos, DOCINADE, Apartado 223-21001, Alajuela, San Carlos, Ciudad Quesada, Costa Rica; 2 Instituto
Tecnológico de Costa Rica, Campus Tecnológico Local San Carlos, Escuela de Agronomı́a, Apartado 223-21001, Alajuela, San Carlos, Ciudad Quesada, Costa Rica; *Author
for correspondence: Tel.: +506 2401 3022; fecheverria@itcr.ac.cr

BioTechniques 71: 581–586 (December 2021) 10.2144/btn-2021-0057

First draft submitted: TBC; Accepted for publication: 23 September 2021; Published online: 12 October 2021

ABSTRACT
The lack of knowledge about biological communities residing in soils, especially those in tropical regions, represents a constraint to manage-
ment practices to take advantage of the ecological services provided by soil microbiota to agroecosystems. One of the complexities derived
from describing biological diversity in such tropical conditions comes from the methods used to isolate microorganisms without altering the
composition of the sample. The goal of this study was to establish a protocol for adequate soil sampling and environmental DNA extraction
from a tropical region in Costa Rica. We present an up-to-date protocol optimized for tropical soils which improves sevenfold the amount of DNA
extracted without significantly affecting the 260/280 and 260/230 ratios compared with commercially available kits and standard protocols.

METHOD SUMMARY
Soils from cultivated pineapple fields and adjacent forests were sampled and stored in cold and 96% alcohol to study and optimize an eDNA
extraction protocol. Three protocols were used and one later modified based on higher DNA quantity and quality.

KEYWORDS:
communities • conservation • metagenomics • pineapple • protocol • storage • tropics

Microorganisms have a fundamental role in plant health and ecosystem services in general [1]. The presence and abundance of microor-
ganisms in the rhizosphere had been shown to be associated with soil particle, fertility and even antagonism to pathogenic-to-crops
fungi and bacteria. Furthermore, their contribution to the adequate development of root consortia may directly influence plant health [2].
Considering all cells, genes and metabolites related to the root consortia, the microbiome information can be useful to understand
the intricate network and function in plant–microorganism interaction [3]. Today, high-throughput DNA sequencing technologies and
metagenomic information from environment samples are a useful alternative to traditional isolation and subculture of specific microor-
ganisms in vitro, microscope identification or biological characterization [4]. However, to accurately identify the biodiversity of the soil,
environmental DNA (eDNA) must be obtained without alteration. The real composition of the soil’s microbial diversity needs to be pre-
served unaltered in each sample. This means that sample collection, transport to the laboratory facility and further sample analysis
must be free of contaminates of any source, such as human manipulation or unsterilized instruments. Without taking controls into
consideration, the biodiversity estimation, abundance or taxon classification can be misinterpreted [5].
Several commercial kits are available on the market for eDNA extraction from different sources. However, not all products and kits
are optimized for tropical soils, especially for soils sustaining natural forests or crops that have higher content of humic acids, organic
matter, clays, and mineral components [6]. Although soil composition chemistry and physical properties differ by environments and
seasons in template regions, soil dynamics and usage for crop production in the tropics are still fertile areas of research due to the
rate of constant denaturalization and mineralization processes. Furthermore, soil use practices affect microbial community structure,
function and ecosystem service provision, which refer to the economic and social activities humans exploit during crop production [7].
The functions and capacity of such soil use can be related to crop performance, soil conservation and environment quality. As such,
function and capacity should be determined not only by the physical and chemical composition of soil, but also by its biological functional
networks [8].
Thus, this article serves as a contribution to the study of the biological communities in tropical soils by developing a descriptive
sampling and eDNA extraction method for downstream applications. The overarching goal is to facilitate soil metagenomic studies that
generate parameters to determined the biological quality of soils and eventually lead to better management strategies for sustainable
exploitation of this natural resource.

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Methods
In the following sections, details of the experiments implemented to modify the extraction protocol are described. In summary, soils
from crop land and adjacent forests were used, and soil conservation once obtained from the field was also considered. At the labora-
tory, eDNA modifications were implemented to further validate the best protocol; eDNA samples were then sequenced to verify library
performance.

Soil samples
Seventy-eight total samples from 10 fields were collected in the framework of the project ‘Analysis of Pineapple Biological Communities in
the North Huetar Region, Costa Rica.’ Soil samples were obtained from pineapple crop field and adjacent forests to consider soil use and
organic matter composition for additional studies. Six hundred grams of soil per hectare were obtained by combining three subsamples
of 200 g each, separated by 3 m. Subsamples were collected from the top 20 cm of soil using an Auger Edelman sand driller and carefully
avoiding direct hand contact with the soil to prevent contamination. Nitrile gloves were used during collection, and tools were thoroughly
washed with water and sterilized with ethanol between each use. Samples were collected at pineapple crop fields and adjacent forested
areas, collecting soil within the root system. All samples were packaged in airtight sealable plastic bags, each labeled accordingly and
transported in a cooler with icepacks to the Molecular Biology Laboratory at the Centro de Investigación y Desarrollo en Agricultura
Sostenible para el Trópico Húmedo, Instituto Tecnológico de Costa Rica. Average transit time between collection and storage at the
laboratory was 3–4 h.

Soil conservation
Samples collected in the field were preserved with two methods: (A) stored at -40◦ C or (B) at lab-controlled temperature (24◦ C) sus-
pended in 96% ethanol. For conservation method A, sample bags were placed directly in the freezer until used in eDNA extraction. For
conservation method B, a homogenized subset of 10 g was placed in a Falcon conical centrifuge 50-ml tube and filled with 96% ethanol.
eDNA extraction was tested from the complete solution (soil + alcohol), only soil (centrifuged pellet and alcohol removed) and only
alcohol (decanted alcohol without disturbing the pellet).

eDNA extraction protocols

Three DNA extraction protocols were evaluated. Protocol 1 (P1) correspond to the commercially available DNeasy PowerSoil Pro Kit
(Qiagen, Hilden, Germany), following manufacturer’s instructions [9]. Protocol 2 (P2) corresponds to the method described [10]. Protocol
3 (P3) corresponds to a version of [11] methodology modified by the authors of this article to improve DNA yield. P3 corresponds to
the improved protocol, whereas P1 and P2 are used for comparison purposes. P1, a proprietary technology, relies on a series of lysis,
binding and washing buffers combined with the use of a spin column to extract and purify nucleic acids. According to the manufacturer,
this protocol was optimized for difficult soil samples and can produce up to eightfold higher yield than other commercially available
kits. P2 relies on a salt buffer for lysis, phenol-chloroform-isoamyl alcohol (PCI) for phase separation and a series of cold isopropanol
washes for clean-up and DNA precipitation [10]. Finally, P3 relies on enzymatic digestion combined with CTAB for cell lysis and uses
PEG and isopropanol for DNA cleanup and precipitation [11]. The main modifications to this protocol are summarized in the Figure 1
and include reducing the amount of sample input, shortening the digestion steps, adding a cold incubation step and modifying several
buffers volume and concentration (details are provided in the Results section).

Optimization of eDNA extraction protocol

To achieve higher quality and quantity of extracted DNA, the effects of different DNA extraction modifications were evaluated. Samples
conserved with method A (frozen) were thawed and brought to room temperature before use in any of the DNA extraction protocols.
Samples preserved with method B (ethanol) were used as is. Regardless of conservation method, all samples were homogenized by
vortexing at 2200 rpm for 10 min, using the corresponding lysis buffers and a ceramic bead. In each case, 500 mg of soil or 500 μl
with liquid samples was used per extraction. DNA quantification was performed with a Nanodrop 8000 spectrophotometer (Thermo
Fisher Scientific, MA, USA). For each sample, DNA 260/280 and 260/230 ratios and concentration (ng/ul) were recorded. The clas-
sification variables used for the standardization of the protocol were extraction buffer volume (in microliters [μl]), SDS volume (μl),
chloroform:isoamyl alcohol (CI, 24:1) volume (μl), use and volume (μl) phenol:chloroform:isoamyl alcohol (PCI; 25:24:1), incubation time
(in minutes), centrifugation time (in minutes), centrifugation temperature (◦ C) and use or no use of sodium acetate 3M and NaCl 5M. The
response variables used to evaluate the protocol efficiency were DNA concentration (ng/μl), 260/280 nm absorbance ratio for nucleic
acids and proteins, and 260/230 nm absorbance ratio for nucleic acids, polysaccharides and phenols.
Descriptive statistics (means and standard errors) were obtained to estimate which variation in the protocol gave the best DNA
quality relation (260/280 and 260/230) and the highest concentration. A principal component analysis (PCA) approach was performed
to describe and visualize the interactions between variables and responses. A comparison of means was made using generalized lin-
ear models with an least significant difference–Fisher multiple comparison test in the cases where significant differences were found
(␣ = 0.05). Only Pearson correlations at a significance level ≤0.05 were considered in order to verify associations between soil com-
position and DNA quality or quantification. A fully complete randomized design (CRD) model with factorial arrangement was used to

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 Buffer 750 µl
+ SDS 10% 250 µL
Pellet
Homogenize lncubate Homogenize lncubate
0.5 g de soil 10 min/2200 RPM 65°C/10 min 1 min/2200 RPM 65°C/10 min

Aqueous phase

Centrifuge lncubate
Sediment 1 vol chloroform Homogenize
20 min/14,500 RPM 4°C/10 min
10 s/2200 RPM

Aqueous phase

Protein
Centrifuge Homogenize lncubate
20 min/14,500 RPM Phenolic phase 10 s/2200 RPM -20°C/60 min
1 vol isopropanol
The steps within the gray area
repeat it two more times.

Centrifuge
Centrifuge Discard Homogenize 2 min/14,500 RPM
20 min/14,500 RPM supernatant 750 µl etOH 10 s/2200 RPM

Discard Centrifuge Resuspend Quantify in

supernatant 20 min/14,500 RPM/20°C with H2O Homogenize
spectrophotometer

Figure 1. Established modified protocol for DNA extraction in tropical soils. The figure describes all steps considered and validated to obtain the
modified P3 protocol.

statistically evaluate the response of the variables (modifications) according to the soil conservation. Each analysis contemplates three
biological replications. The program InfoStat/P v.2008 [12] was used for the statistical analysis.

Library preparation & DNA sequencing

To test the viability of the eDNA extracted for microbiome analysis, samples processes with P1 and P3 were used for library preparation
and sequencing. P2 samples were not included due low quality, as described in the next section. 16S Ribosomal RNA gene amplicon
libraries were prepared using the Illumina 16S Metagenomic Sequencing Library Preparation reagents and protocol, following manufac-
turer’s instructions. This commercial kit uses a PCR amplification approach to generate a ∼550-bp amplicon of the V3 and V4 regions
of the 16S ribosomal gene, used for bacteria and archaea taxonomic classification. All library preparation steps and sequencing were
performed by the North Carolina State University Genomic Sciences Laboratory (NC, USA) using the Illumina MiSeq platform. The aver-
age read quality and number of reads produced per extraction protocol are reported. Read counts and per base sequence quality values
were estimated using FastQC v0.11.9.

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Table 1. Mean value of DNA quantity and quality from tropical soil samples, using three extraction protocols.
Protocol Concentration in ␮g/ul (mean ± SE) A260/280 (mean ± SE) A260/230 (mean ± SE)
P1 116.83 ± 41.34 1.39 ± 0.08 0.87 ± 0.10
P2 197.73 ± 132.33 1.28 ± 0.43 0.65 ± 0.46
P3 1408.60 ± 137.03 1.45 ± 0.03 0.95 ± 0.15
SE: Standard error.

Discussion & conclusions

Comparison of DNA extraction protocols
P3 had the highest DNA mean concentrations compared with P1 and P2 (Table 1). Regarding DNA quality, for all protocols, the recorded
values were below the expected purity standard – that is, a 260/280 absorbance ratio of ∼1.8 and 260/230 absorbance ratio of ∼2.0.
However, P3 produced DNA extractions with higher quality ratios and more consistent results.

Standardization of eDNA extraction protocol from [11]

Selecting which modifications to the methodology were best suited was not straightforward. Different permutations were tested (n = 67).
Optimal results, in terms of DNA quantity and quality, were obtained by reducing sample input from 5 to 500 mg; reducing extraction
buffer volume from 10 ml to 750 μl; reducing SDS concentration from 20 to 10% and volume from 2 ml to 250 μl; not using proteinase
K nor lysozyme in the extraction buffer; incubating in a water bath at 65◦ C for 30 min instead of 2 h; adding a 10-min incubation at 4◦ C
step after the chloroform/isoamylalcohol (24:1, v/v) addition step; precipitating the DNA by adding isopropanol rather than 3 M sodium
acetate along with 0.4 volume of 30% (w/v) PEG-8000; and resuspending the dried pellet in pure water rather than TE buffer.
Figure 2 shows a PCA representing the variance and association in which each modification affected DNA quantity and quality.
Almost 84% of the variance of all modifications was explained by the interaction between concentration and quality. The increase of the
concentration was also related with the 260/230 ratio, whereas 260/280 ratio was slightly affected by both parameters. For instance, the
260/280 ratio was shown also to be affected in some proportion by the addition of sodium acetate, increase in incubation time or use of
higher amount of extraction buffer, however, the opposite was observed with an increase of DNA concentration. Centrifuge time, on the
other hand, was shown to have a higher impact on increasing 260/230 ratio and DNA concentration. No other variable was significant
with regard to increased yield or quality.
On the basis of the different permutations tested and PCA, a new protocol, P3, was developed for eDNA extraction from tropical soil
samples (Figure 1). The steps of the protocol are as follows:

Step 1: Add 500 mg of soil sample, 1 g of ceramic beads (0.5 mm diameter), 750 μl of extraction buffer and 250 μl of 10% SDS to a
2-ml microcentrifuge tube.
Step 2: Vortex for 10 min at 2200 rpm.
Step 3: Incubate the tube at 65◦ C for 10 min, vortex for 1 min at 2200 rpm, and incubate for another 10 min at 65◦ C.
Step 4: Centrifuge the homogenized solution at 14,500 rpm for 20 min. Transfer the supernatant into two new labeled microcentrifuge
tubes.
Step 5: Add one volume of chloroform-isoamyl alcohol (CI) to the supernatant in the new tubes and vortex for 10 s at 2200 rpm, then
place the samples in a cold bath to incubate at 4◦ C for 10 min.
Step 6: Centrifuge at 14,500 rpm for 20 min. Transfer the supernatant into a new labeled microcentrifuge tube. Be careful not to disturb
the pellet.
Step 7: Add one volume of cold isopropanol to the supernatant in the new tubes, mix by inversion for 10 s and incubate at -20◦ C for
1 h.
Step 8: Centrifuge at 14,500 rpm for 20 min, discard the supernatant and wash the pellet with 750 μl of 75% ethanol. Mix by inversion
and then centrifuge for 1 min at 14,500 rpm. Repeat this step twice.
Step 9: Let the pellet dry at room temperature. This allows for any residual ethanol to evaporate. The pellet is then suspended in 50 μl
of ultra-pure distilled water.

Comparison of soil conservation methods

Two methods for tropical soil conservation were compared. eDNA was extracted from samples 2 months after collection. Significant
differences were found between the two preservation methods used (p = 0.0001). Method A (cold storage: -40◦ C) resulted in a higher
concentration of extracted DNA (386.92 ± 169.58 ng/μl). Method B2 (sample stored in 96% ethanol using only the sediment phase)
produced the least favorable results (36.10 ± 23.21 ng/μl) (Figure 3). Quality, in terms of the 260/280 and 260/230 nm absorbance
ratios, was not significantly different between treatments. Average values ranged from 1.34 to 1.40 (260/280) and from 0.64 to 0.78
(260/230). Temperature in the tropics vary greatly during the day, especially when working in open fields. These results remark on the
importance of keeping a proper cold chain from sample collection to laboratory storage. Failure to keep the samples cold significantly

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 5.00

SDS (ul)
2.50 PCI 1st
PCI 2nd

Centrifuge (min)
CI (ul)
260/230
CP 2 (14.6%)

ng/ul
NaCI (ul)
0.00 260/280
Sodium acetate (ul)

Extraction buffer (ul) Incubation (min)

Centrifuge (˚C)
-2.50

-5.00
-5.00 -2.50 0.00 2.50 5.00
CP 1 (29.2%)

Figure 2. Principal component analysis showing the association of protocol modifications and their impact in eDNA concentration and quality. Yellow
dots represent the variable parameter, and blue dots represents the sample analyzed. The figure does not represent the protocol but specific changes
made to the original protocol to improve the recommended P3.

reduces DNA yield. However, if cost, accessibility or distance between collection site and laboratory prohibit the use of cold storage,
then the recommendation would be to use a 96% ethanol solution as a preservative and perform the extraction from the liquid phase
(i.e., ethanol and suspended particles). DNA yield will be low but may be enough for certain applications such as PCR-based identification
of specific taxa.
Soil chemical composition may affect the DNA extraction process and must be taken into account by adapting the extraction buffers
solutions. For example, DNA in an acid solution is insoluble and fragments, therefore, a pH of 8 must be attained using salts, such as
Tris and NaCl, in the buffer solutions [11]. Also the presence of CaCl2 during cell lysis is important because it prevents oxidation of humic
substances, which generate quinones that bind to the DNA and prevent amplification [11]. Chloroform-isoamyl-alcohol has the ability to
separate organic compounds (proteins, phenols, lipids and polysaccharides) from DNA, allowing for phase separation and high-quality
DNA isolation [13]. The buffer chemistry in this study was optimized to favor adequate cell lysis and DNA preservation while preventing
humic acids and other contaminants [14].
The incubation time that generated the higher concentrations and DNA quality in this study was 30 min. Extended incubation periods
have resulted in improved performance in other studies [15,16]. However, in our case, a shorter incubation combined with mixing by vortex
twice reduced the homogenizing time while still effectively reducing protein activity. Centrifugation at 4◦ C was adequate to prevent
further enzyme activity, which may degrade the DNA.

DNA sequencing
eDNA from samples extracted with P3 produced, on average, 138,756.00 ± 38,434.40 sequence reads, whereas eDNA from samples
extracted with P1 only produced, on average, 58,954.60 ± 40,755.71 sequence reads. Regarding per base sequence quality, values were
reported using a Phred score scale, where values above 20 (or a base call accuracy of 99%) were consider acceptable. Both P1 and

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500
386 a

400
Concentration (ng/µl)

300

156 b

200
86 b

36 b
100

A B1 B2 B3
0
Cold -40˚C 96% ethanol Sediment + 96% Sediment
ethanol
-100 Method of preservation

Figure 3. Concentration (ng/μl) (mean ± standard error) of DNA extracted from a tropical soil, using two conservation methods. Means with the same
letter have no significant differences between them according to least significant difference–Fisher (p < 0.05).

P3 produce sequence read with acceptable per base quality average values. However, P3 eDNA extractions did have a higher, although
not statistically significant, mean value (32.13 ± 3.93) compared with P1 (29.40 ± 5.96). More important, considering that only the
DNA extraction protocol differed between samples – that is, same soil samples, library preparation and high-throughput sequencing
conditions – P3 extractions resulted in a significant increase in data yield (p < 0.001, 2.4-fold increment). These differences in data yield
are crucial to guarantee an adequate representation of the taxonomic composition of each sample [17].

Future perspective
Metagenomics studies in tropical regions will dramatically increase in the coming years. Lack of knowledge about soil diversity and
ecological functioning in crop production–soil use interactions will benefit from discovering microbiota networks related to beneficial
and pathogenic dynamics. Our eDNA extraction protocol can enhance the recovery of such data from organic and humic-rich soils, which
gradually can be used in other soil types and related studies. Soil diversity, taxonomic composition and probable association with soil
usage, will be presented in a different study.

Summary points
• The most efficient method for soil sample preservation, in terms of DNA extraction potential, was method A (cold storage at -40◦ C).
• The optimized eDNA extraction protocol (P3) obtained the highest DNA concentration (ng/μl). While the 260/280 and 260/230 ratios were
below the optimal range, the extracted DNA proved adequate for library preparation and high-throughput sequencing. Furthermore, P3
produces on average the most reads per sample, without compromising per base sequence quality.

Author contributions
F Echeverrı́a-Beirute: substantial contributions to the conception and design of the experiment; acquisition, analysis and interpretation of
data; and drafting of the original manuscript. M Carvajal-Chacon: contributed to laboratory work, data acquisition and analysis. I Varela-
Benavides: substantial contributions to the conception and design of the experiment, as well as drafting and revising the manuscript
for critically important intellectual content. JP Jiménez-Madrigal: substantial contributions to drafting and revising the manuscript for
critically important intellectual content. T Guzmán-Hernández: substantial contributions to the conception and design of the experiment
and final approval of the version to be published.

Financial & competing interests disclosure

Funding was provided by Vicerrectorı́a de Investigación y Extensión, ITCR ‘Analysis of Biological Communities of Pineapple.’ The authors
have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

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Open access
This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit
http://creativecommons.org/licenses/by-nc-nd/4.0/

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