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. DNA Extraction Process involves four parts: 1. Put the cells into a solution. 2. Use enzymes to hydrolyze the cell wall. 3. Use a detergent to break apart the cell membrane. 4. Use 95% ethanol to take out the DNA from the lysate. 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….. Extraction Solutions: 1. Bacterial Suspension Medium – used to make a solution of the cells 2. Lysozyme – enzyme that hydrolyses specific bonds that hold the cell wall 3. 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: 1. DNA 2. Nucleases 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 that digest DNA 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. Answers to Lab Q’s 1. 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. 2. Lysozyme-suspension 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. 3. Protein denature at 60 degrees celsius because it contains fewer hydrogen bonds than DNA. DNA is composed of many nitrogenous base pair and so, it has many more hydrogen bonds. 4. A denatured protein has lost its shape and therefore can no longer function. Nucleases in this lab were denatured in the hot water bath. 6. DNA should attach to the spooling rod at the aqueous-ethanol interface.