Laboratory Operations Toolbox Teacher Guide Biotechnology in Action Description This Biotechnology unit is designed to complement the Toolbox 5 series of laboratory-based units and the scenario chosen follows and adds to each of the units in Toolbox 5. The following table demonstrates the scenario’s relationship to the competency units. Unit relationship to competency Competency Scenario section in Toolbox Step 1. Introduction to the Scenario 1. COM500A Provide information to customers Step 2. Communication with Customer 2. ORG500A Schedule laboratory work for a small team Step 3. Planning for extraction of DNA 3. MAIN500A Maintain and control stocks Step 4. Maintaining the Biotechnology Laboratory 4. TEST506A Apply spectrometric techniques Step 5. Extraction and Quantitation of DNA from Lizard Skin 5. TEST500A Calibrate and maintain instruments Step 6. Calibration and Setting up of Equipment 6. PMLTEST507A Apply Chromatographic and Electrophoretic Techniques Step 7. Running the Gel 1. COM500A Provide information to customers Step 8. Communication of Results. Please also note that this biotechnology unit has the following features and does not purport to be a comprehensive introduction to biotechnology. Features of this unit: A brief introduction to biotechnology in general A brief introduction to some of the concepts of biotechnology An exercise to start the student thinking about biotechnology, and A ‘taster’ only, as there is no in-depth material in this unit. Depending on circumstances you may wish to use this unit as an introduction to biotechnology and/or add additional materials specific to requirements to ‘flesh out’ the unit. Teacher Guide Biotechnology in Action 1 Laboratory Operations Toolbox Teacher Guide Step: 3 Forum: Extraction/purification and amplification of DNA In this step, learners will participate in a discussion forum about the extraction/purification and amplification of DNA. Before participating in the discussion forum the learner needs to access some information. The learner should go to the ROCHE website and obtain information on the extraction of DNA from tissues (eg. go to the World Wide Web Catalogue and choose Web Visit – ROCHE from the list provided). Other sites can then be investigated using a search engine term such as ‘DNA extraction’ to obtain further information, especially alternative methods of DNA extraction. The best sites to look at are company-based (suppliers to the biotechnology industry such as ROCHE) or research based (e.g. university or biotechnology company research information). Look for at least one method of PCR amplification. Forum Discussion When you have completed your Internet search go to the Forum and choose the topic Extraction/purification and amplification of DNA. Tutor Note The discussion could be initiated with questions such as: Why is DNA extracted from cells and tissues? How is it extracted? How is purity determined? How is the DNA amplified? In addressing these questions, the learners should discuss use of different reagents during the extraction and purification process. When discussing purity, an explanation of how protein and RNA contamination is detected should be included. On the question of amplification, an explanation of PCR and the importance of temperature program can be raised. It should also include some discussion of the absolute requirement for cleanliness during PCR. Teacher Guide Biotechnology in Action 2 Laboratory Operations Toolbox Teacher Guide Step: 3 Activity: Assignment - DNA Extraction, Purification and Amplification Questions and Answers Answer the following questions based on your Internet website search for DNA Extraction/Purification and PCR. 1. What is DNA extraction/purification and why is it used? DNA extraction/purification is the method employed to extract DNA from cellular or tissue samples and is used to prepare adequate amounts of DNA of high enough purity for subsequent analysis of the DNA. 2. List one method for DNA extraction sourced from a website, such as the ROCHE site. You should present this as a flowchart listing each of the steps. There is no need to explain the steps. There are a number of methods listed on this site. This assignment is designed to make the student aware of DNA extraction/purification as the first steps in DNA analysis BUT not to make the student an expert in this technique. Look for an answer the clearly lists all the steps in the chosen technique. 3. List one other method of DNA extraction and list the website from which it was obtained. Again, use a flowchart to outline the steps. There are innumerable methods listed on the Internet. This assignment is designed to make the student aware of DNA extraction/purification as the first step in DNA analysis BUT not to make the student an expert in this technique. Look for an answer the clearly lists all the steps in the chosen technique. 4. Which of these methods was the better one and why. Look for a clear indication of the better method. Reasons include: cheaper safer reagents less steps - less complicated less time better yield (quantity) better quality. 5. List one method of PCR and list the website from which it was obtained. Again, use a flowchart to outline the steps. There are innumerable methods listed on the Internet. This assignment is designed to make the student aware of PCR as the amplification step in DNA analysis BUT not to make the student an expert in this technique. Look for an answer the clearly lists all the steps in the chosen technique. Teacher Guide Biotechnology in Action 3 Laboratory Operations Toolbox Teacher Guide Step: 6 Activity: Assignment - Agarose Gel Electrophoresis Questions and Answers Answer the following questions using SOP: Agarose Gel Electrophoresis as a guide. 1. When setting up the agarose gel apparatus, including pouring the gel, name three important factors that have a direct bearing of the success of the run. In this case success means a gel with clearly separated and non-distorted bands. Correct set up of apparatus No leaks Correct strength of agarose Samples placed into well correctly Correct buffers used Correct running conditions - voltage and time Agarose prepared correctly - fully dissolved Agarose allowed to set fully Tape removed from ends of casting plate Gel stain added. 2. Can you calibrate the gel apparatus itself? Explain your answer. No, because there are no mechanisms for calibration and nothing that can be adjusted. 3. Can you calibrate the gel itself to account for small variations between separate gels? Explain your answer. Yes, the use of a DNA marker allows calibration of the run, as the sizes of the DNA fragments in the samples are determined from the known sizes of the DNA fragments in the ladder. 4. Name three safety concerns when doing agarose gel electrophoresis. Ethidium bromide is a mutagen Biosafety issues arising from the DNA samples Electricity Burns form molten agar General chemical safety from buffer constituents UV light damage to eyes and skin. 5. Given what you know about the basics of electrophoresis, why do you think that the concentration of agarose may be varied from 1 - 3%? The concentration of agarose is adjusted to suit the size of the fragments being analysed. Smaller DNA fragments require a higher strength agarose for effective resolution and larger DNA fragments require a lower strength agarose. Teacher Guide Biotechnology in Action 4 Laboratory Operations Toolbox Teacher Guide Step: 8 Question and Answers: Correctly filled in form Laboratory Test Sheet - Lizard DNA Samples Date: x/1/2004 Customer Sample Numbers: Lizard 120, Lizard 121, Lizard 122, Lizard 123, George, Fred Sample Description: Lizard skin Sample Type: DNA Sample Volume: N/A - sheet of skin Customer Sample Number Integrity of Sample Sample Accepted/Rejected for testing. Lizard 120 Lizard 121 Lizard 122 Lizard 123 George Fred All samples are intact and suitable for analysis. All samples accepted, none rejected. Client Details: Australian Reptiles Pty Ltd 5 Big River Road SimuTown CIV 0313 Contact: Dr Jake Crawford, Head breeder 9551-2106 Integrity of Samples: Details of any rejected samples are listed here: .................................................. .................................................. .................................................. Tests Required/Results (Bold and underline testing required): Parentage Sex Determination Sequencing Other TEST Results: Sample Identification Lizard 120 Lizard 121 Lizard 122 Lizard 123 Test Result - Paternity George George George George Test Result - Sex Male Female Female Female Checked by: Student name Teacher Guide Biotechnology in Action 5