What is the best method other than using kit to isolate DNA from fiber crops? Any experience and/or tips on how to improve genomic DNA production from fiber crop samples? Which plant part is the best source? How much DNA can be isolated?
Thank you.
Khaled Salem
Grind the texture of 100 g of cotton, fixed in liquid nitrogen, until a homogeneous fine powder is formed in the mortar, adding 100 ml of a solution containing 1 ml of phenol on top of it and crumbling again until a uniform mass is formed. The resulting homogenate is shaken with 100 ml of phenol. The finished mixture is centrifuged at a speed of 20 minutes 6000 ayl/min (10000 g) at 0°C. This time, 3 layers are formed in the centrifuge test tube; at the bottom of the test tube is phenol, in the middle of which there is protein and ground fibrous plant tissue, and the upper part contains DNA aqueous layer, the middle layer is taken and extracted with a "a" solution as above. This operation is continued until there is no DNA left on the water floor. After that, the residue of the plant tissue is extracted again with the above solutions "on" and "v". DNA in solution is determined based on the formation of precipitate when 96% alcohol is poured into a certain amount of extract.
Here are some tips for isolating high quality genomic DNA from fiber crops:
• Use young leaves as the best source of DNA. Leaves contain actively dividing cells and more DNA. Older leaves tend to be more fibrous and contain inhibitors.
• Grind the leaves in liquid nitrogen instead of a mortar and pestle. This helps break down the tough fibers more efficiently. You can grind 1-2 grams of leaves at a time.
• Use a cetyltrimethylammonium bromide (CTAB) based method. CTAB helps disrupt cell membranes and bind inhibitors. Use 2% CTAB and adjust the NaCl concentration to 0.35 M. CTAB works best for fiber-rich plants.
• Include beta-mercaptoethanol in the buffer. This helps reduce oxidized phenols and salts that can inhibit DNA precipitation and shearing. Use 0.2% beta-mercaptoethanol.
• Precipitate the DNA with isopropyl alcohol instead of sodium acetate. This helps remove more contaminants and salts, resulting in higher DNA yields from fibrous tissue.
• Re-suspend the DNA pellet in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) or water instead of ethanol. This helps dissolve the DNA more efficiently without degradation.
• DNA yields can range from 50-200 micrograms per gram of starting leaf tissue depending on the species and age of leaves. You may need to pool DNA from multiple extractions to get enough for applications like sequencing, genotyping, etc.
• Check the DNA integrity on agarose gels to ensure high molecular weight, intact strands before using for downstream applications. Degraded DNA will not perform well.
• Optional: treat the CTAB-precipitated DNA with RNase A to remove any trapped RNA before re-suspending the pellet in TE. This can improve DNA purity.
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