Bacterial Transformation with “glowing” DNA (GFP on a plasmid)

Pre-Lab (Please answer in your lab notebook)
Note – You may need to review some genetics notes from last semester.
3. Why would we want to put DNA into bacteria? 4. How can we tell DNA is in the bacteria once we put it there? 5. What is a plasmid? 6. What is ampicillin?
Materials for each group (students should work in groups of 4):

Tube of plasmid DNA (tube labeled DNA)
One tube of E. coli bacteria (on ice) 2 LB agar plates with selection (ampicillin) (1 and 2 black stripes) Materials to share:
Water bath at 42°C

Procedures & Procedure Questions: (Please answer the questions in your notebook)
1. Pipette the 20 l of DNA solution from your DNA tube into your E. coli bacteria tube and label the tube with your initials.
2. Put tubes on ice for 5 minutes. Why do you think we put the tubes on ice? (Answer in your notebook)
3. In the meantime, each group should get one LB agar plate and two LB agar + ampicillin plate (one with one
stripe one with two stripes). Write your initials, period and your lab station number on your plates. You will be
plating bacteria with DNA on an LB agar plate and on an LB agar + ampicillian pate. Mark these three plates with
the DNA number on your tube, your group members initials and class period. Where is the best place to label
your plates? What is the control you are conducting? (Answer in your notebook)

4. Put tubes directly from ice into 42°C water bath for 50 seconds. What do you think heating the tubes does?
(Answer in your notebook)

5. Put tubes directly from water bath onto ice for 2 minutes.
6. Add 250 l (first notch on the large transfer pipet) of LB broth into 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?
(Answer in your notebook)
7. Add100l (first notch on the small transfer pipet), from your tube onto your LB agar plate and 100 l onto each of your
LB agar + ampicillin plates.
8. Spread the solutions on the plates using a sterile Q-tip (spread LB agar plate first then LB agar + ampicillin plate with one
stripe, and last LB agar + ampicillin plate with two stripes. Be careful not to stab the agar. --Why is it important to spread
the solutions onto the plates in this order?
(Answer in your notebook)
9. Put your plates in a 37°C incubator for 24 hours. Why 37°C?
10. What do you expect to grow on each of the plates? (Answer in your notebook in the chart below)

LB agar + ampicillin
LB agar + ampicillin
Do you expect to see any
(1 black stripes)
(2 black stripes)
difference in bacterial
growth on the plates?

bacteria +

Challenge for Day 2:
What is ampicillin and why do you think we used it in some of the plates?
What do you think the bacterial growth would have looked like on each type of plate (LB agar, LB agar + ampicillin) if you
had added no DNA?

Day 2:
Data Questions:

1. What do you see on your plates?
2. Now look at your plates with UV light. What do you see?
Fill in the table with your data -
What do you see on each type of plate?


LB agar + ampicillin
LB agar + ampicillin
(1 black stripes)
(2 black stripes)
What kind of
growth and
did you see?
What does this
tell you about

Analysis Questions: (Answer in your notebook)
1. What does the DNA allow the bacteria to do?
2. If you observed a difference in the two LB agar + ampicillin plates, hypothesize cause for this difference? How would you
test your hypothesis?
3. What are some potential sources of error in this lab?
4. How might you avoid some of these sources of error?


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