BIO231: Practical: Plant DNA Isolation using CTAB method

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. Once the tissue has been sufficiently ground, it can

then be resuspended in a suitable buffer, such as CTAB (Hexadecyl trimethyl-

ammonium bromide).

CTAB

One of the most commonly used methods to extract DNA from plants uses the

ionic detergent cetyltrimethylammonium bromide (CTAB) to disrupt membranes

and chloroform that separates contaminants into the organic phase and nucleic acid

into the aqueous phase.

Chloroform denatures protein. Isopropyl alcohol precipitates out the DNA.

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.

 Materials
CTAB buffer
Microfuge tubes (Eppendorf tubes)
Mortar and Pestle
70 % Ethanol (ice cold)
65 °C water bath
Chloroform
Isopropanol

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
1 g PVP 40 (polyvinyl pyrrolidone 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 121.1g of Tris base in 800mL of H2O. Adjust pH to 8.0 by adding 42mL
of concentrated HCl. Adjust the volume to 1L with H2O.
To make a 0.5x working solution, do a 1:10 dilution of the concentrated stock.
Procedure
1. Grind 200 mg of plant tissue to a fine paste in approximately 500 μL of CTAB
buffer.
2. Transfer CTAB/plant extract mixture to a microfuge tube.
3. Incubate the CTAB/plant extract mixture for about 15min at 65°C in a
recirculating water bath.
4. After incubation, spin the CTAB/plant extract mixture at 14000 rpm for 10 min
to spin down cell debris.
5. Transfer the supernatant to clean microfuge tubes.
6. To each tube add 250 μL of chloroform and mix the solution by inversion.
7. After mixing, spin the tubes at 14000 rpm for 10 min.
8. Transfer the upper aqueous phase only (contains the DNA) to a clean microfuge
tube.
9. To each tube add 250 μL of isopropanol.
10. Invert the tubes slowly several times to precipitate the DNA.
11. Centrifuge the sample at 14,000 rpm for 10 min.
12. Remove supernatant.
13. Pellet was washed with 70 % ethanol, dried and resuspended in 50 µL of water
containing DNase free RNase A and stored at -20°C.