Dr. Alexander W.

Kena
Dept. of Crop and Soil Sciences,
KNUST, Kumasi.

CS 558: Plant Biotechnology

Methods for genomic DNA extraction

There are numerous methods currently available for extraction of total genomic DNA from
both plant and animal sources including microbes. The choice of method to use depends on
important factors such as the required quantity and molecular weight of the DNA, the purity
required for downstream applications, and the time and cost involved. Regardless of the type
of method used, one can identify common steps associated with all the methods presently
available for DNA extraction from cellular components. Basically, all methods start with
disruption and lysis of cell or tissue, followed by removal of proteins and other contaminants,
and finally recovery of DNA. Each method seeks to maximize DNA recovery and quality
(purity) by removing inhibitors or components that degrade DNA such as nucleases. The
most commonly used extraction procedures can be categorized as:
A. Organic-based methods (Phenol-chloroform)

B. Non-organic-based methods (proteinase K and salting out)

C. Anion-exchange methods

D. Chelex (ion exchange resin) extraction

E. Silica-based methods

A. Organic extraction methods (Phenol-chloroform isolation)

This method utilizes organic solvents to remove proteins and other contaminants from cell
lysates. Cells are lysed using a cell lysis buffer containing a detergent (SDS or CTAB), after
which cell lysates are then mixed with phenol and/or chloroform with iso-amyl alcohol to
remove proteins and other contaminants upon centrifugation. The iso-amyl alcohol is added
to prevent foaming. The cell lysis buffer usually contains EDTA (Ethylenediaminetetraacetic
disodium salt), which is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor
for Dnase nucleases. If the Mg2+ is bound up by EDTA, nucleases are inactivated which
protects the DNA. Also, RNAse is added to remove RNA. The correct salt concentration and
pH of the isolation buffer should be used to ensure DNA remains in the aqueous phase after
centrifugation. This procedure takes advantage of the fact that, deproteinization is more
efficient when two different organic solvents are used instead of one. DNA in the aqueous
phase is precipitated using either ice-cold isopropanol or 95-100% ethanol. DNA precipitates
in the presence of moderate concentrations of monovalent cations such as Na+, and it’s
recovered by centrifugation and redissolved in an appropriate buffer such as TE (Tris-EDTA
Buffer) or deionized water for storage. Pelleted DNA is then washed with 70% ethanol to
remove salt residues. Genomic DNA obtained using this method may contain residual phenol
and/or chloroform, which can inhibit enzyme reactions in downstream applications such as
PCR. The method also requires the use of toxic chemicals that must be disposed of with care
and in accordance with hazardous waste guidelines. In addition, this technique is almost
impossible to automate, making it unsuitable for high-throughput applications.

Materials

CTAB buffer
Microfuge tubes
Mortar and Pestle
Liquid Nitrogen
Microfuge
Centrifuge
Absolute Ethanol (ice cold)/Isopropanol (ice-cold)
70 % Ethanol (ice cold)
7.5 M Ammonium Acetate
55- 65 oC water bath
Chloroform:Iso Amyl Alcohol (24:1)
Water (sterile)

Agarose
6x Loading Buffer
1x TBE solution
Agarose gel electrophoresis system
Ethidium Bromide solution
CTAB buffer 100ml

2.0 g CTAB (Hexadecyl trimethyl-ammonium bromide)

10.0 ml 1 M Tris pH 8.0
4.0 ml 0.5 M EDTA pH 8.0 (EthylenediaminetetraAcetic acid Di-sodium salt)
28.0 ml 5 M NaCl
40.0 ml H2O
1g PVP 40 (polyvinyl pyrrolidone (vinylpyrrolidine homopolymer) Mw 40,000)
Adjust all to pH 5.0 with HCL and make up to 100 ml with H2O.

1 M Tris pH 8.0

Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust pH to 8.0 by adding 42 ml of

concentrated HCL. Allow the solution to cool to room temperature before making the
final adjustments to the pH. Adjust the volume to 1 L with H2O. Sterilize using an
autoclave.

5x TBE buffer

54 g Tris base
27.5 g boric acid
20 ml of 0.5M EDTA (pH 8.0)
Make up to 1L with water.
To make a 0.5x working solution, do a 1:10 dilution of the concentrated stock.

1% Agarose gel

1 g Agarose dissolved in 100 ml TBE

Procedure

- Grind 200 mg of plant tissue to a fine paste in approximately 500 μl of CTAB buffer.
- Transfer CTAB/plant extract mixture to a microfuge tube.
- Incubate the CTAB/plant extract mixture for about 15 min at 55o C in a recirculating
water bath.
- To each tube add 250 μl of Chloroform: Iso Amyl Alcohol (24:1) and mix the
solution by inversion. After mixing, spin the tubes at 13000 rpm for 1 min.
- After incubation, spin the CTAB/plant extract mixture at 13000 rpm for 10 min to
spin down cell debris. Transfer the supernatant to clean microfuge tubes.
- Transfer the upper aqueous phase only (contains the DNA) to a clean microfuge
tube.
- To each tube add 50 μl of 7.5 M Ammonium Acetate (optional) followed by 500 μl
of ice-cold absolute ethanol (or ice-cold isopropanol).
- Invert the tubes slowly several times to precipitate the DNA. Generally, the DNA can
be seen to precipitate out of solution. Alternatively, the tubes can be placed for 1 hr at
-20 o C after the addition of ethanol to precipitate the DNA.
 - Following precipitation, the DNA can be pipetted off by slowly rotating/spinning a tip in
the cold solution. The precipitated DNA sticks to the pipette and is visible as a clear thick
precipitate. To wash the DNA, transfer the precipitate into a microfuge tube containing
500 μl of ice cold 70 % ethanol and slowly invert the tube. Repeat. ((alternatively the
precipitate can be isolated by spinning the tube at 13000 rpm for a minute to form a
pellet. Remove the supernatant and wash the DNA pellet by adding two changes of ice
cold 70 % ethanol )).
- After the wash, spin the DNA into a pellet by centrifuging at 13000 rpm for 1 min.
Remove all the supernatant and allow the DNA pellet to dry (approximately 15 min). Do
not allow the DNA to over dry or it will be hard to re-dissolve.
- Resuspend the DNA in sterile DNase free water (approximately 50-400 μl H2O; the
amount of water needed to dissolve the DNA can vary, depending on how much is
isolated). RNaseA (10 μg/ml) can be added to the water prior to dissolving the DNA to
remove any RNA in the preparation (10 μl RNaseA in 10ml H2O).
- After resuspension, the DNA is incubated at 65o C for 20 min to destroy any DNases that
may be present and store at 4o C.
- Agarose gel electrophoresis of the DNA will show the integrity of the DNA, while
spectrophotometry will give an indication of the concentration and cleanliness.

DNA integrity and purity test

DNA integrity test showing intact DNA DNA absorbance at 260 nm wavelength
and DNA smear

B. Non-organic-based methods (proteinase K and salting out)

This method does not use organic reagents such as phenol or chloroform. Instead, proteins are
first digested using proteinase K enzymes, after which they are removed by salting out with high
concentrations of LiCl, or potassium acetate or ammonium acetate. Upon centrifugation, the
aqueous phase is pipetted into a new tube to pellet DNA using ice-cold isopropanol. Precipitated
DNA is then washed with 70% ethanol, dried under vacuum and re-suspended in TE buffer for
Page 4 of 6
storage. If salts are not adequately removed, problems could occur in downstream applications
such as RFLP procedure due to alteration of DNA mobility. Sometimes repeated isopropanol
precipitation is required to improve on the the purity of the pelleted DNA.

C. Anion-exchange methods
Solid-phase anion-exchange chromatography is based on the interaction between the negatively
charged phosphates of the nucleic acid and positively charged surface molecules on the substrate.
DNA binds to the substrate under low-salt conditions, impurities such as RNA, cellular proteins,
and metabolites are washed away using medium-salt buffers, and high-quality DNA is eluted
using a high-salt buffer. The eluted DNA is recovered by alcohol precipitation and is suitable for
all downstream applications. Anion-exchange technology completely avoids the use of toxic
substances, and can be used for different throughput requirements as well as for different scales
of purification.

D. Chelex (ion exchange resin) extraction

Chelex 100 is an ion exchange resin that is added as a 5% solution (wt/vol). Chelex consists of
styrene divinylbenzene copolymers containing paired iminodiacetate ions that act as chelating
groups in binding polyvalent metal ions such as magnesium (Mg2+). By removing the Mg2+ from
the reaction, nucleases are inactivated, and the DNA is protected. A 5% solution of Chelex is
added to the cell lysate and incubated at 56 oC for 30 minutes. This step is used to remove
contaminants and inhibitors. The sample is then heated at 100 oC for 8 minutes. This causes the
DNA to be denatured as well as disrupting membranes and destroying cellular proteins. The tube
containing the Chelex is centrifuged, the resin is pelleted, the supernatant containing the DNA is
then removed. The Chelex extraction process denatures double stranded DNA and yields single
stranded DNA, and thus cannot be used for the RFLP procedure. It is advantageous for PCR-
based typing methods because it removes inhibitors of PCR and can be done in a single tube,
which reduces the potential for laboratory-induced contamination.

E. Silica-based methods
The DNeasy protocol is a silica-based resin method that is based on a simple bind-wash-elute
procedure. The procedure ensures selective binding of DNA and separate nucleic acids within

Page 5 of 6
certain pore size parameters. A variety of modified silica gel surfaces and optimized binding
buffers are used to obtain maximum discrimination between nucleic acids during adsorption and
washing steps. Nucleic acids are adsorbed to the silica gel membrane in the presence of
chaotropic salts, which remove water from hydrated molecules in solution. Polysaccharides and
proteins do not adsorb and are removed. After a wash step, pure nucleic acids are eluted under
low- or no-salt conditions in small volumes, ready for immediate use without further
concentration. The methods is fast, rapid and devoid of toxic chemicals such as phenol and
chloroform.

Page 6 of 6