BIOTECH Project, University of Arizona
Bacterial Transformation
Name:
Period:
Bacterial Transformation with Mystery DNA
Some questions to get you thinking about today’s lab:
What can we use DNA for?
How can DNA be put into bacteria?
Why would we want to put DNA into bacteria?
How can we tell DNA is in the bacteria once we put it there?
What is a plasmid? What is ampicillin?
Materials for each group (students should work in groups of 4):
Tube of mystery plasmid DNA (tubes numbered 1, 2, 3 or 4) 1 tube of LB broth
One tube of E. coli bacteria (on ice) micropipette
1 LB agar plate micropipette tips
1 LB agar plate with ampicillin for DNA (1 black stripes) sterile transfer pipette
Q-tips or innoculation loops
Materials to share:
Water bath at 42°C
ice
trash containers/biohazard waste bag
UV lights
Protocol:
1. Using the syringe pipettor and a sterile tip, pipette the DNA solution from your numbered DNA tube into your E. coli bacteria tube and label the tube according to your DNA number (1, 2, 3, 4). Also mark your tube so that you will recognize it compared the other groups.
2. Put tubes on ice for 5 minutes. Why do you think we put the tubes on ice?
3. In the meantime, each group should get one LB agar plate and one LB agar + ampicillin plate. You will be plating bacteria with DNA on an LB agar plate and on an LB agar +ampicillian pate. Mark these two plates with the DNA number on your tube, your group members initials and class period. Also label which plat contains Ampicillian. Where is the best place to label your plates? What is the control you are conducting
4. Put tubes directly from ice into 42°C water bath for 50 seconds. What do you think heating the tubes does?
5. Put your tube directly from the water bath onto ice for 2 minutes.
6. Add all of LB broth into the transformation tube by tipping the LB tube upside down onto the transformation tube and flicking the LB tube. Incubate at room temperature or in your hand for 10 minutes. What is the LB broth for? Why would you want the cells to be as warm as your hand?
7. Add an equal amount of LB Broth, DNA and E coli. from your transformation tube onto your LB agar plate and the LB agar + ampicillin plate by pipetting an equal number of drops on each plate with the transfer pipet.
8. Spread the solutions on the plates. Be careful not to stab the agar. The same Q-tip can be used for both plates as long as it is kept sterile (don't touch it to anything!).
9. Put your plates in a 37°C incubator for 24 hours. Why 37°C?
Challenge for Day 2: What is ampicillin and why do you think we used it in some of the plates?
What do you expect to grow on each of the plates?
LB agar / LB agar + ampicillin(1 black stripe) / Do you expect to see any difference in bacterial growth on the two plates?
bacteria + DNA / Would expect to see
bacterial lawns
What do you think would have grown on these plates if no DNA had been added to these bacteria?
LB agar / LB agar + ampicillin(1 black stripe) / Do you expect to see any difference in bacterial growth on the two plates?
bacteria (without DNA)
Day 2:
What do you see on your plates?
E. coli are normally white to off white, do any of the bacteria have a different color??
Now look at your plates with UV light. What do you see?
Fill in the table with your data and the class data-
what does each group (#1, 2, 3, 4) see on each type of plate?
Mystery DNA(number) / LB agar / LB agar + ampicillin
(1 black stripes) / Based on the phenotype,
what is the DNA?
#1
#2
#3
#4
What does each DNA type (1, 2, 3, 4) allow the bacteria to do? How many genes are in each plasmid and what does each gene produce?
1