AP Laboratory: Microbes in Action Bacterial Transformation & Gel Electrophoresis

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Giles Murphy
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1 AP Laboratory: Microbes in Action Name: Bacterial Transformation & Gel Electrophoresis Introduction In this laboratory you will use some basic tools of molecular biology to gain an understanding of some of the principles and techniques of genetic engineering. In the first part of the lab, you will use antibiotic-resistance plasmids to transform Escherichia coli. In the second part, you will use gel electrophoresis to separate fragments of DNA for further analysis. 1. Log onto the Pearson LabBench web page at 2. Select Lab 6 Molecular Biology 3. Follow the instructions carefully on each screen be sure to review all animations. 4. Answer the lab packet questions as you proceed through the virtual labs. 5. There are 2 lab activities: Part I - Transformation in Bacteria and Part II Gel Electrophoresis 6. Be sure to conclude both labs in your Lab NB this will be collected for a grade! Instructions for concluding the virtual lab are at the end of this packet! 1. Read the Introduction section. What are the TWO goals of this lab? 2. Read Key Concepts I: Bacterial Transformation. What is bacterial transformation? a. What gene are we going to attempt to introduce into our bacterial strain? b. What will happen to our bacteria strain if we are successful? 3. Read Bacterial Colonies. What species of bacterial are we using for this experiment? 4. Read E. coli Bacteria. Where is E. coli bacteria commonly found? L. Carnes / J. Maloney AP Biology Microbiology Adapted from Pearson: LabBench Activities

2 a. How does E. coli reproduce? How rapidly can this occur? b. Describe the chromosomal genome of E. coli. 5. Read Plasmids. What are plasmids? a. How are plasmids used in genetic engineering? b. What does the amp R gene confer resistance to in E. coli? c. What happens to E. coli cells that do not carry the amp R gene? d. In order to transform cells...what do we need to do first? 6. Read Competent Cells. Describe what this concept means. a. What processes are used to make cells competent?

b. In what phase of bacterial growth is it easiest to make cells competent? 7. Read Design of Experiment I. Make notes of some important safety procedures: 8. Read Transformation Procedure.

3 b. In what phase of bacterial growth is it easiest to make cells competent? 7. Read Design of Experiment I. Make notes of some important safety procedures: 8. Read Transformation Procedure. After you've familiarized yourself with the procedure as a whole, take a closer look at each stage. Select steps 1 4 and 6 to see what is going on at the cellular level. THE VERY LAST A Closer Look screen shows an animation of the transformation procedure...watch IT! There are basically SIX steps to follow to correctly transform bacteria. Describe these steps IN ORDER below: a. b. c. d. e. f. 9. The figures below show the events that take place during transformation of an E. coli cell. Type in the letters to indicate the correct order in which the events occur.

4 10. Read Analysis of Results I. Draw AND describe the four possible outcomes for this experiment in the space provided below. a. What does it mean if there is a lawn growth of cells? b. What does it mean if there are individual colonies of growth on the agar? c. What does it mean if nothing is growing on the agar? 11. Read Label the Results of Your Experiment. After incubation, the following plates were removed from the incubator. All but part of one label have been removed so that you must now use your understanding of this laboratory to make new labels for each plate. Based on the PHYSICAL APPEARANCE of each plate, select the appropriate label for the plates. DESCRIBE THIS BELOW...do NOT simply write A, B, C, or D! For example...plate I should be labeled because.

5 a. Plate I: b. Plate II: c. Plate III: d. Plate IV: 12. Take Lab Quiz I. You may write your responses below: Question #1 Question #2 Question #3 Question #4 Question #5 Question #6 13. Read Key Concepts II: Electrophoresis. What are restriction enzymes? What is the source of restriction enzymes? a. How do restriction enzymes work? b. In what way did the discovery of restriction enzymes make genetic engineering possible?

6 c. What is our goal in this part of the laboratory? 14. Read How do Restriction Enzymes Work?. Click on each of the cut options and watch the animation. Describe below how restriction enzymes accomplish cutting up DNA into fragments: a. What does palindromic mean? 15. Read Cutting DNA with Restriction Enzymes. Briefly describe how this is accomplished: 16. Read Gel Electrophoresis. Click the run tab to view the animation. What is gel electrophoresis? a. What affects the direction of movement in gel electrophoresis? b. What affects the rate of movement in gel electrophoresis?

7 c. How is DNA charged? In which direction will it move? d. Which fragments will move the farthest? Why? e. How is the actual length of each fragment of DNA measured? 17. Read Design of Experiment II. In order to understand each step, you will need to progress through the next few screens. BE SURE TO CLICK ON AND WATCH ALL OF THE AVAILABLE ANIMATIONS! Describe below the steps involved in the procedure: a. Prepare the Gel: b. Obtain Prepared DNA Samples: c. Load Samples into Gel: d. Separate Fragments by Electrophoresis: e. Stain DNA and Measure Fragments: f. Determine Fragment Sizes:

18. Read Analysis of Results II. How do researchers determine the size of DNA fragments produced with particular restriction enzymes? 19. Read Making a Standard Curve for HindIII DNA Fragments.

8 18. Read Analysis of Results II. How do researchers determine the size of DNA fragments produced with particular restriction enzymes? 19. Read Making a Standard Curve for HindIII DNA Fragments. Describe how fragment size is determined by this method...then use this information to complete the measured distance chart. 20. Read Making a Standard Curve. Watch the animation. Describe the following: a. Semilog Graph Paper:

9 21. Read Practice Problem I. Place your answers in the chart below. 22. Read Practice Problem II. Place your answers in the chart below. 23. Take Lab Quiz II. You may write your responses below: Question #1 Question #2 Question #3 Question #4 Question #5 Question #6 Question #7 Question #8

10 Analysis & Conclusions This section should be completed in your lab NB: 1. Title of Lab #1 Transformation in Bacteria a. Intro/Background for Transformation Lab b. Materials/Methods - briefly describe the procedure for transforming bacteria in the lab (from the Transformation Procedure screen). c. Results draw & describe the online lab results into your NB (from the Analysis of Results I screen). d. Conclusions what did you learn? e. NOTE: drawn and labeled diagrams should be included for all sections when appropriate! 2. Title of Lab #2 Gel Electrophoresis a. Intro/Background for Electrophoresis Lab b. Materials/Methods - briefly describe the procedure for running a gel electrophoresis in the lab (from the Design of Experiment II screen). c. Results draw & describe the online lab results into your NB (from the Making a Standard Curve for HindIII DNA Fragments screen). d. Conclusions what did you learn? e. NOTE: drawn and labeled diagrams should be included for all sections when appropriate!

Bacterial Transformation Lab - pglo

Bacterial Transformation Lab - pglo Name: Date: Pre-Lab Score: Lab Overview: In this investigation, you will gain an understanding of the techniques of culturing E. coli bacteria and transforming using