Name: Biology Lab: Mr. Zimmerman Date: Period: I. Objectives Virtual Bacterial Identification Lab To familiarize you with the science and techniques used to identify different types of bacteria based on their DNA sequence To work together with a partner to reveal the materials and procedures involved in a professional laboratory designed to identify bacterial DNA. II. Process Skills Comprehending Interpreting Analyzing III. Materials Laptop Notebook HHMI website (http://www.hhmi.org/biointeractive/vlabs/bacterial_id/index.html) IV. Background 1. Not long ago, DNA sequencing was a time-consuming, tedious process. With readily available commercial equipment and kits, it is now routine. The techniques used in this lab are applicable in a wide variety of settings, including scientific research and forensic labs. Basic Steps Prepare a sample from a patient and isolate whole bacterial DNA. Make many copies of the desired piece of DNA. Sequence the DNA. Analyze the sequence and identify the bacteria. 2. The segment of DNA used for identifying bacteria is the region that codes for a small subunit of the ribosomal RNA (16S rrna). We will refer to this piece as 16S rdna. Different bacterial species have unique 16S rdna sequences. The identification relies on matching the sequence from your sample against a database of all known 16S rdna sequences. V. Procedure 1. Throughout each exercise, the window will display information explaining what you are doing. All the interactions, however, will be done inside the graphic window to the left. The small white box below the graphic will give you specific instructions on what objects to click on. 2. Below is a list of each of the six parts of the lab. Work through each carefully, reading the information to the right of the procedure.
Part 1: Bacterial Sample Preparation VI. Analysis and Conclusions-Part A (1) For each of the terms below, explain the exhibited ROLE or PURPOSE as observed in the virtual laboratory process. Use the reading and your observations as a guide in constructing meanings behind each term. Part 1: Bacterial Sample Preparation: (a) Gloves: (b) Inoculating loop: (c) Nutrient culture: (d) Microcentrifuge tube: (e) Orange tray: (f) Digestive enzymes: (g) Digestive buffer: (h) Micropipette: (i) Biohazard container: (j) Heated water bath: (k) Centrifuge: (l) Counterbalance: (m) Supernatant:
(n) PCR tube: (a) PCR master mix solution: (b) Green-capped positive control bottle: (c) Deionized water bottle: (d) PCR machine: (a) Microconcentrator column: (b) Yellow-capped buffer bottle: (c) PCR product: (d) Ice bath: (e) Centrifuge: (f) New collection tube: (g) Buffer solution: (h) Blue-capped distilled water: (i) Green and blue strip tubes: (No terms) (a) Automatic Sequencer:
(a) Genbank: (b) BLAST: (c) NCBI site: VI. Analysis and Conclusions-Part B (1) What are primers? (2) What types of patient samples are used for the purpose of identifying possible pathogens? (3) What does PCR do, how does it work, and why is it useful? (4) How do you separate the desired DNA from all others? (5) How does an automatic DNA sequencer work? (6) How is it possible to use a DNA sequence to identify bacteria?
(7) According to your findings, which species of infectious bacteria did your patient suffer from? (8) Finally, provide a brief synopsis of the major features that were involved behind each of the following parts of the lab. What were the overall objectives or key points that were recognized during each stage? Part 1: Bacterial Sample Preparation