DNA Isolation From Plant Tissue - Procedure
DNA is isolated from the rest of the cell contents by taking advantage of the different chemical and physical properties of the molecules found within the cell. Let's take a moment to review cellular structure. Where is DNA located within the cell? What other chemical compounds are found in a cell? How is the cell compartmentalized?
Each organism is different and the cells of each kind of organism often contain special compounds, which must be extracted away from the DNA. In the case of Echinocereus cacti, the cells of the tissue contain MUCILAGE!!! Mucilage (often used as glue!) is a very sticky, jelly-like substance whose function in the plant cells is not known for certain, but it is thought to act as an antifreeze. Chemically, mucilage is a highly complex polysaccharide - very big, linear molecules, which have the ability to form large complexes with other large molecules such as DNA. The mucilage is contained within the cytoplasm of the cell, while the DNA is contained within the nucleus (and within mitochondria and chloroplasts). To isolate DNA from the tissue of Echinocereus, the mucilage must be prevented from coming into contact with the DNA and complexing with it. In order to accomplish this, the DNA must remain within the nuclei while the mucilage is released from the cell and then poured off. This procedure requires the use of some dangerous and toxic chemicals, so we won't attempt to do this in a high school classroom. However, we would like you to have the experience of isolating DNA. Therefore, we will perform a DNA isolation from the tissue of a plant that is not so difficult to work with.
In addition to removing carbohydrate compounds like mucilage, the DNA isolation procedure must also separate the DNA from compounds like lipids (fats) found in cell membranes and proteins (enzymes and structural components) which are found throughout the cell. The DNA isolation procedure makes use of the fact that lipids and proteins are soluble in chloroform and that the DNA is soluble in water (aqueous) solutions.
DNA Isolation Protocol
1. Mechanically break open the cell walls and membranes by grinding the tissue in a coffee grinder. The plant tissue should be frozen before grinding and ground in the coffee grinder with dry ice. Keeping the tissue frozen prevents enzymes in the cells from breaking down the DNA. [CAUTION: Do not touch the dry ice as it may cause "cold burns."]
2. Place the pulverized tissue into a 50 ml centrifuge tube containing about 10 mL of CTAB buffer solution. The buffer solution contains compounds which disable the enzymes which will break down the DNA and also a compound called CTAB which will combine with the polysaccharides and proteins and chemically alter them. Mix the solution well to suspend the cellular material. Incubate the solution at 60°C for at least 30 minutes.
3. Pour an equal volume (the same amount as is already in your tube) of chloroform into the 50 mL tube. "Extract" the solution by tilting or rocking the tube gently back and forth (if you mix the solution vigorously you may break your DNA molecules!). Chloroform is an organic compound and all of the cellular compounds which are soluble in chloroform (lipids, proteins) will be dissolved into the chloroform. DNA is not soluble in chloroform and will remain dissolved in the aqueous (water) layer. [CAUTION: Chloroform must be used in a fume hood so that vapors will not be inhaled.]
4. Spin the solution in the centrifuge for 5 minutes. The gravitational forces in the centrifuge will completely separate the two phases according to density. Chloroform is very dense and will be the bottom layer, while the water layer is less dense and will be on the top.
5. Transfer the supernatant (the aqueous layer containing the DNA) to a separate tube with a pipette.
6. Add 2/3 volume of ice-cold isopropanol to the supernatant.
The DNA dissolved in the supernatant will not be soluble in the ice-cold alcohol
and will start to precipitate out of solution over a period of a few minutes.
After a few minutes, gently rock the tube back and forth (if you rock
or shake your tube hard you will break your DNA molecules). The DNA should
appear as whitish strands to clear jelly-like strands which will float in the