Isolation of Plant Genomic DNA

Aim: To demonstrate the isolation of plant genomic DNA using CTAB method
Principle:
All plant DNA Extraction protocol Comprise of the basic steps of disruption of the cell wall, cell
membrane & nuclear membrane to release the DNA into solution fallowed by precipitation of DNA
while ensuring removal of contaminating biomolecule such as the proteins, polysaccharides, Lipids
secondary metabolites.

Introduction
1. Lysis of cell membrane:

The first step of the DNA extraction is the rupture of the cell and nucleus wall. For this purpose. the
homogenized sample is first treated with the extraction buffer containing EDTA, Tris/HCT and CTAB.
The lysis of the membranes is accomplished by the detergent (CTAB) contained in the extraction
buffer. When the cell membrane is exposed to the CTAB extraction buffer, the detergent captures
the lipids and the proteins allowing the release of the genomic DNA. In a specific salt (NaCl)
concentration, the detergent forms an insoluble complex with the nucleic acids. EDTA is a chelating
component that among other metals binds magnesium Magnesium is a cofactor for DNAse. By
binding Mg with EDTA, the activity of present DNAse is decreased. TrisHCI gives the solution a pH
buffering capacity (a low or high pH damages DNA). After the cell and organelle membranes (such
as those around the mitochondria and chloroplasts) have been broken apart, the purification of
DNA is performed
2. Extraction
In this step, polysaccharides, phenolic compounds, proteins and other cell lysates dissolved in the
aqueous solution are separated from the CTAB nucleic acid complex. Under low salts concentration,
the contaminants of the nucleic acid complex do not precipitate and can be removed by extraction
of the aqueous solution with chloroform. The chloroform denatures the proteins and facilitates the
separation of the aqueous and organic phases. Once the nucleic acid complex has been purified,
precipitation can be accomplished.
3. Precipitation:
In this final stage, the nucleic acid is liberated from the detergent. For this purpose, the aqueous
solution is first treated with a precipitation solution comprising of Sodium acetate, which
precipitates the nucleic acid. Under these conditions, the detergent, which is more stable in alcohol
than in water, can be washed out, while the nucleic acid precipitates. The successive treatment
with 70% ethanol allows an additional purification, or wash, of the nucleic acid from the remaining
salt.
 Materials:

 Plant samples (leaf, callus etc.)

 Liquid nitrogen
 Sterile pestle and mortar
 Sterile spatulas
 Water bath set at 65 C
 Sterile Eppendorf tubes and desired reagents

Chemicals:
DNA isolation stocks (100 ml)
SI Components Stock Concentration/ Working concentration For
No Volume 100 ml
.
1 Extraction buffer 1M Tris HCl, pH 8.0 100 mM Tris HCl, pH 8.0 10 ml Tris HCL(1
(CTAB) 05 M EDTA, pH 8.0 20 mM EDTA, pH 8.0 M)
5 M NaCl 1.4 M NaCl 4 ml EDTA (0.5 M)
28 ml NaCl(5 M)
2 Potassium acetate 60.0 ml 5M potassium 5 M (60 -80 μl) _
(200 ml) acetate
11.5 ml Glacial acetic
acid
28.5 ml H2O
3 Sodium acetate adjust pH to 5.2 with 2.5 M ( 200 ml) _
(200 ml) glacial acetic acid and
make up the volume
5 T10E1 (TE 1 M Tris HCL 10 mM Tris HCL 1 ml Tris HCL(1 M)
Buffer) 0.5 M EDTA 1 mM EDTA 0.2 ml EDTA (0.5 M)
98.8 dd H2O
6 RNase _ 10 mg/ml (5 μl)
7 Ethanol 100% 70% 70 ml Ethanol
+ 30 ml dd H2O

8 PVP – Polyvinylpyrrolidone - Pinch of PVP

9 Phenol : Chloroform : Isoamyl Alcohol (25:24:1)

or
Chloroform : Isoamyl Alcohol (24:1)
10 Isopropanol
General Protocol :

Plant Genomic DNA Extraction:

1. Take 100 mg tissue, homogenate to powder with liquid nitrogen and transfer the powder to sterile
Eppendorf tube.

2. Add 1 ml of Extraction buffer (preheated at 65 C for 15 min.) to homogenate, mix vigorously and
incubate in water bath at 65°C for 1 hr.

3. Bring down the sample temperature to RT, add 667μl chloroform: Isoamyl alcohol (24:1), mix
gently by inverting for a period of 15-20 min.

4. Spin at 10,000 rpm at 4 °C for 10 min, collect the supernatant, and add 2/3 ml of cold isopropanol.
Mix gently to precipitate the nucleic acid. Spin for 5-10 minutes.

5. Wash with around 500μl of 70% ethanol. Decant and dry the pellet at room temperature. Dissolve
in 50 µl of 0.1X TE+ Rnase (100µg/ml). Incubate hr at 37° C.

Procedure: CTAB Method

1. Fresh emerging leaves were harvested and wrapped in the foil paper.

2. These were immediately taken to the laboratory and kept in the refrigerator at -20°C
to retain the freshness of the material.

3. The leaves were vigorously rinsed in distil water to remove particles on leaf surface

4. About 200 mg each sample was gently ground into autoclaved pestle and mortar with 1 ml CTAB,
extraction buffer (Pre heated at 65°C for at least 10 minutes).
NOTE : For Mature leaf us of Liquid N is advised

5. Transfer the crushed sample into 2ml Eppendorf tube

6. A volume of 4 μl B-mercaptoethanol was added to each tube which was then mixed thoroughly by
gently mixing.

7.Samples were incubated in a water bath at 65°C for at least 30 minutes (1 hour is
preferable)

8. Samples were retrieved from the water bath after incubation and allowed to return to
room temperature for 5-10 minutes.

9. An equal volume of chloroform: isoamylalcohol (24:1) te.. 1 ml to each tube was added for
extraction. This was mixed gently by continuously inverting the tubes for 5 min.

10. Samples were centrifuged at 13000 rpm for 13min at 27°C to separate the phase
11.An equal volume of chloroform: isoamylalcohol (24:1) e. 1 ml to each tube was added for
extraction. This was mixed gently by continuously inverting the tubes for 5 min.

12. Samples were centrifuged at 13000 rpm for 15min at 27°C to separate the phase.

13. Top light green colour aqueous phase was transferred to new 1.5 ml micro centrifuge tube, along
with 0.75 volume chilled isopropanol (i.e. that is for 1 ml aqueous phase, 750 μl chilled isopropanol
added) to precipitate the DNA & add 60 μl of Potassium Acetate.

14. Samples were mixed gently by continuous inversion and kept at -20°C overnight to
recover the DNA pellets:

15. The samples were centrifuged at 6000 rpm for 8 min at 4°C

16. Carefully, the supernatant was discarded without disturbing the DNA pellet.

17. The pellets were washed in 70 % ethanol and air dried until ethanol evaporates completely from
the samples. This was facilitated by inverting tubes on tissue paper.

18. Pellet dried and dissolved in TE, of about 50 – 100 μl depending on the size of the pellet and
treated with 2 μl RNase (10 mg/ml). Samples were incubated at 37°C for 1 hour.