1 Biology 2250 Transformation laboratory Prior to lab: Discuss how to use micropipettes. Discuss how to use microcentrifuge balance. Discuss how to spread bacteria with alcohol and glass rods. Bacteria are a type of model organism. Click on the link below to find out what are examples of other model organisms. In this laboratory, you will use bacteria to learn how to perform a transformation which is the process of introducing DNA into bacterial cells. We will be introducing a plasmid as the foreign DNA. You will be given a plasmid that has a gene for Ampicillin resistance. If the bacteria take up the plasmid, then they will be resistant to that antibiotic. Antibiotic Resistance is simply where the bacterium can withstand an antibiotic, like our bacterium to Ampicillin. But, if we introduce the plasmid with a resistance gene for ampicillin, it will be resistant to that antibiotic and no others. Different plasmids have different antibiotic resistance genes. If the bacteria take up a different plasmid, they may have resistance to a different antibiotic. We are going to take our bacterium and mix it with the plasmid DNA that contains a resistance gene to Ampicillin and then we will plate the bacteria on agar plates that have either the antibiotic ampicillin or kanamycin mixed into the agar. By the end of this laboratory, the student should understand: What a transformation is. How a transformation is different from a transduction. How is transformation different from conjugation. What are the different parts of a plasmid. What is the purpose of antibiotics in a transformation. Why the untransformed and transformed bacteria grew differently on the kanyomycin and ampicillin plates. Look at the picture of the plasmid below. What are the different parts?
3 Do you think our bacteria that did NOT undergo the transformation procedure will or will NOT grow on a plate that contains Kanamycin or Ampicillin? What do you think will happen if our bacterium takes up the plasmid by transformation; should they grow on Ampicillin or Kanamycin? You will perform 2 transformations and have 2 untransformed controls Tube 1: Untransformed bacteria plate on LB+ Kanamycin Tube 2: Untransformed bacteria plate on LB+ Ampicillin Tube 3: Transform bacteria with pglo - plate on LB + Kanamycin Tube 4: Transform bacteria with pglo - plate on LB + Ampicillin You will also have another tube as a control to make sure the bacteria grow fine Tube 5: Untransformed bacteria plate on LB *untransformed means that they DO NOT get plasmid but they do go through the entire procedure. Which of these groups should grow on Kanamycin? Which of these groups should grow on Ampicillin?
4 Supplies needed/group 2 LB + Kan plates 2 LB + Amp plates 1 LB plate 1 LB plate with HB101 bacteria streaked on them. 2 tubes of pglo plasmid (labelled tube 3 and 4) Protocol 1. Label microcentrifuge tubes 1, 2,3, 4 and 5. The contents are described above. Also get 1 LB plate, 2 LB + Amp Plates and 2 LB + Kan plates, 1 starter plate with bacteria (these should be in a stack with a rubber band). Put your name or initials on them. 2. Pipet 250 L of transformation solution (0.05 M CaCl2) into tubes 1-5 and place the tubes on ice. 3. Transfer 2-3 colonies from your starter plate into the microcentrifuge tubes labeled 1-5 that you put the transformation solution in. You will use toothpicks to pick the colonies. Use aseptic technique. Keep the starter plate because you will need it at the end of the experiment. 4. Pipet 5 L of the plasmid into tubes 3 and 4 only. Do not add any plasmid to the tubes labeled 1, 2 and 5. These tubes do not get any plasmids. 5. Incubate ALL five of the tubes on ice for 10 minutes. 6. Transfer ALL five of the tubes directly from the ice into a 42 degree water bath for exactly 50 seconds. Then immediately put back on ice for 2 minutes. 7. Remove ALL five of the tubes from the ice and pipet 250 L of LB broth into each tube using sterile technique. Incubate the tubes at room temperature for 10 minutes. 8. Mix the tubes by inverting. 9. Take 100 L of each sample and spread it on the appropriate plate using the glass rod and spreader. I have shown below the five groups and what plate they go on:
5 Tube 1: Untransformed bacteria plate on LB+ Kanamycin Tube 2: Untransformed bacteria plate on LB+ Ampicillin Tube 3: Transform bacteria with pglo - plate on LB + Kanamycin Tube 4: Transform bacteria with pglo - plate on LB + Ampicillin Tube 5: Untransformed bacteria plate on LB 10. Place your plates in the 37 degree incubator.