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STUDENT GUIDE: Bacterial Transformation with 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 plasmid DNA |
1 tube of LB broth
|
| One tube of E. coli bacteria (on ice) |
micropipette
|
| 1 LB agar plate |
micropipette tips
|
| 2 LB agar plates with ampicillin for DNA (1 and 2 black stripes) | small transfer pipette |
| Q-tips or inoculation loops |
large transfer pipette
|
Materials to share:
Water bath at 42°C
ice
trash containers/biohazard waste bag
UV lights
Protocol:
1. Pipette the DNA solution from your numbered DNA tube into your E. coli
bacteria tube and label the tube 1 or 2
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 two LB
agar + ampicillin plates (one with one black stripe and one with two black stripes). Write your initials and your section number on
your plates. You will be plating bacteria with DNA on an LB agar plate
and on both LB agar +ampicillin plates. 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 DNA tubes directly from water bath onto ice for 2 minutes.
6. With a large transfer pipette, add 0.25 ml LB broth into your DNA tube
( bring solution to the first mark on the pipette.) Incubate at room temperature
for 10 minutes. What is the LB broth for?
7. With the small transfer pipette, pipette 0.10 ml from your DNA tube onto
your LB agar plate and 0.10 ml 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 black stripe, and last LB agar + ampicillin
plate with two black stripes. Be careful not to stab the agar.
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 think the bacterial growth would have looked like on each
type of plate if you had added no DNA?
What do you expect to grow on each of the plates?
|
LB agar
|
LB agar + ampicillin
(1 black stripes) |
LB agar + ampicillin |
Do you expect to see any difference in bacterial growth on the two plates? | |
|
bacteria + DNA |
Day 2:
What do you see on your plates?
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?
|
Type of agar plate
|
LB agar
|
LB agar + ampicillin |
LB agar + ampicillin
(2 black stripes |
|
What kind of growth and phenotype(s) did you
see? |
|||
|
What does this tell you about the DNA? |
What does the DNA allow the bacteria to do?
If you observed a difference in the two LB agar + ampicillin plates, hypothesize cause for this difference? How would you test your hypothesis?
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BIOTECH Project Last Modified March 8, 2002 Nadja Anderson, Ph.D. |