I am working on DNA methylation and would want to extract DNA from maize leaves and roots. If you have protocols please direct me to them.
Hi Sylvain
Any CTAB method works very well for plant tissue. They can by modified but all originates from this article : A rapid DNA isolation procedure for small quantities of fresh leaf tissue, By Doyle and Doyle. Avaible here on ResearchGate.
Best Britta
You could try this protocol, which is very useful for plant total DNA extraction.
3% CTAB DNA extracion protocol
(modified from Zeng et al. 2002, ACta Botanica Sinica 44; 694-697)
1. washing out polysaccharides with CTAB-free buffer from homogenate.
2. increasing the percentage of CTAB in extraction medium.
3. using high concentration salts pior to DNA precipitation with isopanol.
Hello Sylvain.
Below is a method which might help you;
Collect 10cm long piece of fresh leaf material
Freeze in liquid N and grind to a fine powder.
Add 800 μl DNA Extraction Buffer and mix well (vortex).
Add 5 μl RNaseA (10mg/ml); keep on 37ºC water bath for 30 minutes.
Add 800 μl Phenol:Chloroform:Isoamylalcohol (25:24:1) and shake well
Centrifuge for 10 minutes at max speed.
Transfer the upper phase (about 650 μl) to a fresh tube.
Add 650 μl Chloroform and mix gently.
Centrifuge for 10 minutes at max speed.
Transfer the supernatant (about 450 μl) to a fresh tube.
Add 45 μl 3M Sodium Acetate (pH = 4.8) and 450 μl Isopropanol.
Mix gently. DNA will precipitate. Stored @-20ºC for 30 min.
Centrifuge for 10 minutes at max speed.
Pour off the supernatant. Wash pallet with 600 μl of 70% Ethanol. Mix well.
Centrifuge for 10 minutes at max speed.
Pour off the supernatant. Dry pallet on the rack for 30 minutes in the hood.
Resuspend in 100 μl of deionized H2O. Mix gently. Stored @-20ºC.
Solutions:
DNA Extraction Buffer (for 1 Lit.)
33ml 30% Sarkosyl (1% of N-Lauroyl Sarcosine)
1% SDS
12.1 g. Trizma base (Tris base)
5.8 g. NaCl
3.2 g. Na2EDTA
pH= 8.5 by HCl
Phenol:Chloroform:Isoamylalcohol
250ml redistilled phenol
240ml chloroform
10ml Isoamylalcohol
TE
10 mM Tris
1 mM EDTA
pH= 8.0
All the best
Prince
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