Pacing/Teacher's Notes Investigation #8: Transformation Click on - PDF document

Slide 1 / 31 Slide 2 / 31 New Jersey Center for Teaching and Learning AP BIOLOGY Progressive Science Initiative This material is made freely available at www.njctl.org and is intended for the non-commercial use of Investigation #8 students and teachers. These materials may not be used for any commercial purpose without the written Biotechnology: Bacterial Transformation permission of the owners. NJCTL maintains its website for the convenience of teachers who wish to make their work available to other teachers, participate in a virtual professional learning community, and/or provide access to course materials to parents, students and others. Summer 2014 www.njctl.org Click to go to website: www.njctl.org Slide 3 / 31 Slide 4 / 31 Pacing/Teacher's Notes Investigation #8: Transformation Click on the topic to go to that section · Pacing/Teacher's Notes · Pre-Lab · Guided Investigation · Independent Inquiry Return to Table of Contents Slide 5 / 31 Slide 6 / 31 Pacing Teacher's Notes General Reference to Lab procedure adapted from College Board AP Biology Day (time) Activity Notes Description Unit Plan Investigative Labs: An Inquiry Approach Teacher's Manual Day 1 (HW) GE Day 9 Pre-lab Background Prepare plates for tomorrow HW Be sure to review instructions for Click here for CB Guided Transformation your plasmid, and add any Day 2 (80) GE Day 10 Practice & Plating necessary steps. AP Biology Incubate plates overnight Teacher Manual Calculating Day 3 (40) Analysis Transformation GE Day 11 Efficiency Independent Transformation Day 4 (40) GE Day 12 Inquiry & Plating Calculating Transformation Independent Day 5 (40) GE Day 13 Inquiry Efficiency & Discussion Day 6 (20) Assessment Lab Quiz GE Day 14

Slide 7 / 31 Slide 8 / 31 Pre-Lab Question/Objectives How can we use genetic engineering techniques to manipulate heritable information? In this lab we will: · Demonstrate the universality of DNA and its expression. · Explore the concept of phenotype expression in organisms. · Explore how genetic information can be transferred from one organism to another. · Investigate how horizontal gene transfer is a mechanism by which genetic variation is increased in organisms. · Explore the relationship between environmental factors and gene expression. · Investigate the connection between the regulation of gene expression and observed differences between individuals in a population of organisms. Return to Table of Contents Slide 9 / 31 Slide 10 / 31 Pre-Lab Questions Pre-Lab Questions Read the background information and answer the following Read the "Getting Started" and answer the following questions in questions in your lab notebook. your lab notebook. 1. What causes mutations in bacteria? Can mutations affect 1. Some bacteria are naturally resistant to antibiotics, but others are plasmids? not. How could you use two LB/agar plates, so E. coli , and some ampicillin (an antibiotic) to determine how E. coli cells are affected by 2. What is the function of plasmids in bacteria? ampicillin? 2. What would you expect your experimental result to indicate about 3. Do cells take up more plasmids in some conditions and less in the effect of ampicillin on the E. coli cells? Do you think that exposure others? to ampicillin will cause the E. coli cells to evolve resistance to ampicillin? Why or why not? 3. How will you be able to tell if host E. coli cells have been genetically transformed? Slide 11 / 31 Slide 12 / 31 Safety Guided Investigation When working with a culturing bacteria, it is important not to introduce contaminating bacteria or fungi into the experiment. Because these microorganisms are ubiquitous, i.e., you can find them everywhere - on fingertips, bench tops, lab tables, etc. - you must avoid these contaminating surfaces. When working with inoculation loops, bulb pipettes, micropipettes, and agar plates, do not touch the tips of them (or in the case of agar, the surface itself) or place them directly onto contaminating surfaces. Be sure to wash your hands before beginning the procedure and after - and cover your sneezes. Do not eat, drink, apply cosmetics, or use personal electronic devices in the work area. Return to Table of Contents

Slide 13 / 31 Slide 14 / 31 Materials Guided Practice Your Workstation · Transformation solution (CaCl 2 ) Step 1 Familiarize yourself with sterile technique before · E. coli starter plate · Microcentrifuge tubes and holder beginning the experiment. · 2 LB agar plates · Container of crushed ice · 2 LB/amp agar plates · Marking pen Step 2 Label one closed microcentrifuge tube "+plasmid" and · LB nutrient broth · Lab notebook the other tube "-plasmid". Label both tubes with your group's · Sterile inoculation loops number, and place them in the microcentrifuge tube holder. · 100-1000 microliter bulb pipettes · 1-10 microliter micropipettes with sterile tips Step 3 Carefully open the tubes and using a 100-1000 microliter bulb pipette with a sterile tip, transfer 250 microliter of Common Workstation the ice cold transformation solution into each tube. · DNA plasmid · 42 o C water bath and thermometer Step 4 Place both tubes on (into) the ice. · 37 o C incubator · 20 microliter adjustable-volume micropipette · 10% household bleach · Biohazardous waste disposal bags · Masking or lab tape Slide 15 / 31 Slide 16 / 31 Guided Practice Guided Practice Step 5 Use a sterile inoculation loop to pick up a single colony Step 8 Using a 1-10 microliter micropipette with a sterile tip, of bacteria from your starter plate. Be carful not to scrape off transfer 10 microliters of plasmid solution directly into the E. coli any agar from the plate. Pick up the "+plasmid" tube and suspension in the "+plasmid" tube. Tap tube with a finger to immerse the loop in the CaCl 2 solution at the bottom of the tube. mix, but avoid making bubbles in the suspension or splashing Spin the loop between your index finger and thumb until the the suspension up the sides of the tube. Do not add the entire colony is dispersed in the solution. Appropriately discard plasmid into the "-plasmid" tube! the loop. Step 9 Incubate both tubes (+plasmid and -plasmid) on ice for Step 6 Use a new sterile 100-1000 micropipette to repeatedly 10 minutes. Make sure the bottom of the tubes make contact pulse the cells in solution to thoroughly resuspend the cells. with the ice. Place the tube back on the ice. Step 10 While the tubes are sitting on ice, label each of your Step 7 Using a new sterile inoculation loop, repeat Steps 5 and agar plates on the bottom (not the lid). 6 for the "-plasmid" tube. Slide 17 / 31 Slide 18 / 31 Guided Practice Guided Practice Step 11 Following the 10-minute incubation at 0 o C, remove the Step 13 Remove the holder containing the tubes from the ice tubes from the ice and "heat shock" the cell in the tubes. It is and place on lab counter. Using a 100-1000 microliter critical that the cells receive a sharp and distinct shock! Make micropipette with sterile tip, transfer 250 microliters of LB nutrient broth to the "+plasmid" tube. Close the tube and gently sure the tubes are closed tightly! Place the tubes into a test tube holders float, and dunk the tubes into the water bath, set at tap with your finger to mix. Repeat with a new sterile micropipette for the "-plasmid" tube. 42 o C, for exactly 50 seconds. Make sure to push the tubes all the way down in the holder so that the bottom of the tubes with the suspension makes contact with the warm water. Step 14 Incubate each tube for 10 minutes at room temperature. Step 12 When the 50 seconds have passed, place both tubes back on ice. For best transformation results, the change from Step 15 Use a 10-1000 microliter micropipette with sterile tip to transfer 100 microliters of the transformation (+plasmid) and the 0 o C to 42 o C and then back to 0 o C must be rapid. Incubate the tubes on ice for an additional two minutes. control (-plasmid) suspensions onto the appropriate LB and LB/ amp plates. Be sure to use a separate pipette for each of the four transfers.