DNA Extraction Lab Step by step procedure that weakens the outer boundaries of a cell and lyses it to release the DNA for future study. Bacterial cells will be used to learn how to perform this biotech technique. E. coli is the specific type of bacteria to be used. Before Beginning the investigation, watch the link What is Bacteria? to learn about them. Now watch the link E. coli to learn specifically about these cells.

Structure of E. coli

A different strain of bacteria that can be used in this investigation The above is the bacteria used in the DNA extraction lab. Note its Spherical appearance and the color after gram staining. The gram stain specifically stains the cell wall. According to this picture, is it gram positive or negative? Check out the Bacteria Power Point to find out about Gram Positive and Negative Staining…..

Medical Information

DNA Extraction Process involves four parts: Put the cells into a solution. Use enzyme to hydrolyze the cell wall. Use a detergent to break apart the cell membrane. Use 95% ethanol to take out the DNA from the lysate.

Extraction Solutions: Bacterial Suspension Medium – used to make a solution of the cells Lysozyme – enzyme that hydrolyses specific bonds that hold the cell wall ( not used in this lab…why?) SDS – a detergent that lyses the cell by removing the lipids from the cell membrane By adding these solutions to a test tube of bacteria, a lysate is made that contains DNA.

The Lysate Contents of the lysate after shaking, rotating and inverting the tube becomes viscous because the following are added to the suspension: 1. DNA and RNA 2. Nucleases 3. Cytoplasm 4. All types of proteins 5. Fragments of nuclear membrane

The lysate is then placed in a hot water bath set at 60-65 degrees Celsius. Why? Enzymes denature at high temperatures. Nucleases are enzymes which speed up reactions that breakdown nucleic acids. DNA must be protected from the nucleases so the hot water bath destroys the damaging nucleases in the lysate.

DNA Extraction Technique: In this picture, the student used 95% ethanol to cause the DNA to precipitate out of the lysate. DNA is soluble in the lysate but insoluble in ethanol. By drawing the lysate up into the alcohol layer, the DNA comes in contact with the ethanol, thus, becoming visible to the naked eye.

DNA sticks to glass.

Points about the Lab The SDS removes the lipids from the cell membrane. Therefore, it weakens it and disrupts it to release the contents of the cell along with DNA. All DNA extractions require some form of detergent. Lysozyme solution is added before the SDS to break down the cell wall. It hydrolyzes the bonds in the cell wall. Once the cell wall is weakened, the SDS can disrupt the cell membrane. Not all cells require this solution, and only certain types of bacteria require it. Cell wall structure differs in Gram positive and Gram negative type bacteria. Protein denatures at 60 degrees Celsius because it contains fewer hydrogen bonds to hold its structure together compared to DNA. DNA is composed of many nitrogenous base pair and so, it has many more hydrogen bonds. A denatured protein has lost its shape and therefore can no longer function. Nucleases in this lab were denatured in the hot water bath. DNA should attach to the spooling rod at the aqueous-ethanol interface because DNA is insoluble in ethanol and soluble in the lysate.