The four major types of molecules found in a cell are lipids, carbohydrates, nucleic acids and proteins. There are a number of procedures used to extract and purify these molecules. The following procedure is used to illustrate some of the basic principles involved in the extraction of DNA from a cell. In particular, DNA will be extracted from the gram negative bacteria, Escherichia coli.

The general steps in any procedure to extract DNA typically involves suspending the cells in a buffer, breaking open the cells, and then using differential solubility to partially purify the DNA.

Materials: 1.5 ml microfuge tubes; pipettors 250ul, 50 ul; Suspension buffer: 89mM Tris, 1mM EDTA, pH 8.8; Lyse solution: 10% SDS; Precipitation solution: Isopropanol

Procedure:

1. Obtain a plate of nutrient agar upon which E. coli have been grown for between 24-72 hours at 37C.

Pipettor Technique: Slowly depress the plunger on the pipette to the first stop, immerse the tip about ¼ inch into the buffer and slowly release the plunger. The liquid should rise into the pipet tip. Touch the the tip of the pipette to the inside of the microfuge tube and slowly depress the plunger to the first stop and then continue to depress to get the last bit of liquid out of the pipet tip. TRIS is one of several buffers, including phosphate and acetate, that are often used in biological solutions to maintain pH. EDTA is also often used in biological solutions. It acts as a chelator, binding calcium and other ions, which helps inactivate enzymes and destabilize membranes

2. Using a pipettor, pipette 500ul of Tris-EDTA buffer into 1.5ml microfuge tube. If the pipettor is set for 250ul, this procedure should be done twice to deliver the 500 ul.

3. Aseptically transfer, using a loop, approximately ¼ of a loopful of bacteria into the microfuge tube containing the TRIS-EDTA buffer. Mix the bacteria to get them uniformly suspended. If available, use a vortex.

4. Using the 50ul pipettor, pipette 50 ul of 10% SDS into the microfuge tube containing the bacteria. Close the tube and rock it gently, for 30 seconds to mix the solution. Let the solution stand for 3 minutes.

Comment: The solution should become clear and viscous indicating that the bacteria have been broken open and that the DNA has been released. SDS is an anionic detergent that is often used to break open cells. It breaks up lipid membranes and sticks to various molecules, particularly proteins, disrupting their structure.

5. Using a pipettor, add 500 ul of isopropanol to the tube with the now lysed bacteria. If the pipettor only holds 250ul, this procedure should be done twice to deliver the 500 ul. After the addition of the alcohol, close the tube and gently rock the tube for 2-3 minutes to thoroughly mix the solution. The DNA should appear as a whitish cottony mass of material within 2-3 minutes.

Comment: Alcohols such as isopropanol or ethanol are often used to precipitate nucleic acids. The DNA is insoluble in the alcohol solution while many other molecules, including many proteins are soluble in this solution. This is called differential solubility. The DNA could then be removed from the solution, thereby, partially purifying the DNA as a number of contaminating molecules are left behind in the solution.