DNA EXTRACTION AND POLYMERASE CHAIN REACTION TECHNIQUE

DNA EXTRACTION Now that you’ve collected samples of bacteria from the biofilms it is time to extract the DNA from the cells to identify the organisms.

DNA extraction involves several steps and multiple tools DNA extraction involves several steps and multiple tools. Here is a simple explanation of the basic steps. Step 1: The cell walls and membranes of the bacteria must be broken apart. The cell wall is made out of protein and the cell membrane is made out of molecules called lipids (similar to grease on your dirty dishes). Q: What do you use to remove grease from dishes? A reagent called a lysing buffer is added to “lyse” or break the cell membranes, and you guessed right, it is just like the soap you use to wash dishes. Another substance called an enzyme works like a pair of scissors to break up the proteins in the cell walls.

Step 2: Once the cells are broken apart the DNA can be more easily extracted. Fun facts: *DNA is a negatively charged molecule *Most proteins are positively charged. The samples are incubated or heated aid in the process of breaking up the proteins. Many molecules do not like high temperatures and the heat causes the molecules to break into smaller pieces. FUN FACT: * The heating process also breaks up the enzymes you added to destroy the cell wall. This is helpful in removing the unwanted material in the sample, smaller pieces are easier to filter. After incubation, the samples are filtered using a special column that will trap the DNA but allow all the other stuff we don’t need to pass through.

Step 3: Once the samples have been incubated, a centrifuge is used to pull the liquid through the special filter. This is done a couple of times using different solutions. The first time the centrifuge is used, a solution is added to pull everything but DNA through the filter. The second time the centrifuge is used, a solution is used to pull the DNA out of the filter and into a new container called a micro centrifuge tube. Now we are ready to make copies of the DNA using PCR.

Polymerase Chain Reaction (PCR) The final step before sequencing is to make a lot of copies of our DNA sample so that we have enough for the sequencing machine to detect and read. Polymerase Chain Reaction or PCR allows us to target specific DNA segments and quickly make copies. View this short video to see how PCR works. Now try this: Visit this site to carry out a virtual bacteria identification lab. Answer the questions on the next slide as you complete the virtual lab.

Virtual Lab Questions Step 1 Step 2 Step 3 What is the first step in extracting DNA from the bacteria? What part of the cell is involved and what are the chemicals and processes used to facilitate this? What are the four nucleotides added to the master mix? Why are these important? What are the positive and negative controls used in the PCR process? What is the function of a positive and a negative control? Summarize the steps and note the temperatures and molecules involved: During sequencing how are the A, C, T and G’s identified? Step 1 Step 2 Step 3