Isolation of DNA From Plant Material

BIOCHEMISTRY LABORATORY PRACTICE EXPERIMENT 1:

Isolation of DNA from plant material

Suitable sources of nucleic acids are yeast (for RNA isolation) and thymus or spleen (for
DNA isolation). The plant material is in general not as suitable because of the presence of cell
walls and supportive structures that is hard to disrupt without an enzymatic digestion and
using of liquid nitrogen. In the case of soft reaped fruit the cell wall saccharides are
depolymerized as the result of the combined action of several cell wall-modifying enzymes,
acting in both pectic and hemicellulosic fractions allowing the easy homogenization and cells
disruption.
Most important pyrimidine bases are cytosine (Cyt), uracil (Ura) and thymine (Thy). Uracil is
presented almost entirely in RNA, whilst thymine in DNA. Minor bases such as 5-methyl
cytosine and 5-hydroxymethyl cytosine are rarely present. The most important purine bases
are adenine (Ade) and guanine (Gua). A minor purine base is hypoxanthin (Hyp).
The steps in DNA extraction from eukaryotic cells include homogenization of a tissue and
disruption of the cells, followed by precipitation of nucleoprotein and purification. This is
performed either by selective precipitation of proteins and nucleic acids by organic solvents or
adsorption to solid phase.
For each material the process of extraction has to be optimized with respect to specifics of
given material and further application.
In the case of DNA extraction from fresh abundant material, selective precipitation by organic
solvents is sufficient, but in the case of small, ancient or residual samples, or special
applications, the adsorption to solid phase is used due to the higher yield and purity.
In order to disrupt the tissues, various homogenization techniques are used in order to achieve
uniform homogenate in as less time as possible without increasing the temperature. Most
frequently the material is homogenized after freezing by liquid nitrogen in a mortar with
pestle in a buffer containing DNAse inhibitors (usually EDTA is added as chelator of DNAse
metal cofactors), high salt concentration and detergent. Hyperosmotic conditions together
with detergents lead to cells disruption and membranes degradation. Eventually the cell lysate
is further purified from tissue debris by decanting or centrifugation.
DNA from the cell lysate is either adsorbed to solid phase, usually in form of minicolumns or
is precipitated by addition of cold alcohol. For this step is essential to be carried out
immediately after cells disruption. Addition of alcohol and shock cooling leads to decrease of
DNA solubility observable as formation of white precipitate that can be spooled on glass or
wooden stick.
The obtained DNA is still in complex with proteins (nucleoprotein). Addition of organic
solvents non-mixable with water leads to precipitation of the proteins and extraction of
membrane residues to organic phase, while DNA remains in water phase. This can be
repeated several-times followed by precipitation of DNA by cold alcohol.

Material: plant tissue (half of banana), knife, sealable plastic bags, wooden stick, beakers,
calibrated cylinders, microtubes
Chemicals: extraction buffer (1M NaCl, 1g/ml SDS, 1 mg/ml EDTA), chilled isopropanol
(90%)
Isolation protocol:
1. Slice the banana and cut it to small pieces

Using of soft tissue allows us to minimize the homogenization steps.
2. Transfer the pieces in a sealable bag and homogenize it by pressing

During this step the tissue is homogenized.
3. Add the extraction buffer to the bag and pour the mixture into a beaker
The cells are disrupted by hyperosmotic condition and detergents. Addition of EDTA caused
inhibition of DNA-degrading enzymes.
4. Slowly pour the ice-cold isopropanol and observe DNA precipitation

The sock cooling and decreasing the polarity of a solution leads to change of DNA solubility.
The DNA precipitate appears during 2 3 min leaving the solution undisturbed.
5. Use a wooden stick to transfer the precipitate to microtube

Slowly spool the precipitate on a stick and transfer it into a microtube with 1 ml of distilled
water.
6. Measure the absorbance of the preparate at 260

and 280 nm and estimate the DNA concentration
Mix 20 l of DNA sample with water, transfer to a
quartz cuvette and measure absorbance at 260 and
280 nm. The DNA concentration is proportional to
A260, while proteins contamination is proportional
to A280.
In Tab. 1 find the % of nucleic acids (%
nukleovch kyselin) and corresponding F
Set your values to this equation to calculate the
protein contamination:
proteins (mg/ml) = F * A280/d, where d is cuvette

length.
7. Keep the tube for agarose gel electrophoresis

Store the microtube in -20C.
8. After the electrophoresis asses the DNA quality.

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BIOCHEMISTRY LABORATORY PRACTICE EXPERIMENT 1:

Isolation of DNA from plant material

1. Slice the banana and cut it to small pieces

2. Transfer the pieces in a sealable bag and homogenize it by pressing

4. Slowly pour the ice-cold isopropanol and observe DNA precipitation

5. Use a wooden stick to transfer the precipitate to microtube

6. Measure the absorbance of the preparate at 260

proteins (mg/ml) = F * A280/d, where d is cuvette

7. Keep the tube for agarose gel electrophoresis

8. After the electrophoresis asses the DNA quality.

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