Genomic DNA Extraction from Plant

           Mr. Khadga Bikram Angbuhang

                   Department of Microbiology  

GoldenGate Intl’ College

Research officer: LEAD Nepal.

Genomic DNA Extraction from Plant

(pdf version)

Introduction

Plant materials are among the most difficult for high quality DNA extractions. The key is to properly prepare the tissues for extraction. DNA extraction from plant tissue can vary depending on the material used. Essentially any mechanical means of breaking down the cell wall and membranes to allow access to nuclear material, without its degradation is required. For this, usually an initial grinding stage with liquid nitrogen is employed to break down cell wall material and allow access to DNA while harmful cellular enzymes and chemicals remain inactivated. (Liquid nitrogen is difficult to handle and it is dangerous in an open laboratory environment. Alternatively, Cell can be brake without Liquid Nitrogen). Once the tissue has been sufficiently ground, it can then be re-suspended in a suitable buffer, such as CTAB. In order to purify DNA, insoluble particulates are removed through centrifugation while soluble proteins and other material are separated through mixing with chloroform and centrifugation. DNA must then be precipitated from the aqueous phase and washed thoroughly to remove contaminating salts. The purified DNA is then suspended and stored in TE buffer or sterile distilled water. This method has been shown to give intact genomic DNA from plant tissue.

Reagents and Buffers

Extraction Buffer A (EBA)                                                                          Per 100 mL

2% (w/v) hexadecyltrimethylammonium bromide (CTAB)              2.0 g

100 mM Tris (pH 8.0)                                                                         10 mL

20 mM EDTA                                                                                     1 mL

1.4 M NaCl                                                                                         8.2 g

4% (w/v) polyvinylpyrrolidone (PVP)                                               4.0 g

0.1% (w/v) ascorbic acid                                                                    0.1 g

10 mM β-mercaptoethanol (BME)                                                     70 µL

100 mM Tris-HCl (pH 8.0)                                                                 10 mL

Extraction Buffer B (EBB)                                                                          Per 100 mL

50 mM EDTA                                                                                     2.5 mL 100 mM

NaCl                                                                                                    0.6 g 10 mM

β-mercaptoethanol (BME)                                                                  70 µL

TE Buffer                                                                                                       Per 100 mL

1 mM EDTA (Use 0.5 M stock)                                                         50 µL

10 mM Tris (pH 8.0)                                                                          1.0 mL

Other Required Reagents

• 20% (w/v) sodium dodecyl sulphate (SDS)
• 5 M potassium acetate (Stored at –20^o C)
• 3 M sodium acetate (pH 5.2)
• 70% ethanol (stored at -20^o C)
• Absolute isopropanol (stored at -20^o C)
• Water bath of temperature 65⁰ C.

Extraction Procedure

1. Take about 0.3 g of plant tissue and chop in fine pieces.
2. Homogenize with tissue homogenizer in cold condition.
3. Immediately transfer tissue to a 1.5 mL microcentrifuge tube
4. Once the sample is prepared add 300 µL EBA, 900µl EBB, and 100 µl SDS.
5. Vortex and incubate at 65^o C for 10 min.
6. Place tube on ice and add 410 µL cold potassium acetate. Mix by inversion and place tube back on ice for 3 min.
7. Centrifuge at 13,200 rpm for 15 min at 4⁰ C.
8. Transfer 1 mL of the supernatant to a new 1.5 mL microcentrifuge tube, add 540 µL of ice cold absolute isopropanol, and incubate in ice for 20 min.
9. Centrifuge at 10,200 rpm for 10 min and discard the supernatant.
10. Wash the pellet once in 500 µL 70% ethanol.
11. Resuspend the pellet in 600 µL of TE. Add 60 µL 3M sodium acetate (pH 5.2) and 360 µL ice cold absolute isopropanol. Incubate on ice for 20 min.
12. Centrifuge at 10,200 rpm for 10 min and discard the supernatant. (better to wash 2-3 times i.e. repeat 10 and 11 steps).
13. Resuspend the pellet in 50 µL TE and Stored at deep freeze.

(Recommended agar concentration for gel electrophoresis - 1% Agarose gel).

(Protocol modified from Keb-Llanes et al. (2002) Plant Molecular Biology Reporter, 2002).

Reference as: Keb-Llanes, M., Gonzalez, G., Chi-Manzanero, B. and Infanted, D., 2002. A Rapid and Simple Method for Small-Scale DNAExtraction in Agavaceae and Other Tropical Plants. Plant Molecular Biology Reporter, Volume 20, pp. 299a-299e.