BIOTECH Project, University of Arizona
Bacterial Transformation
Bacterial Transformation with “glowing” DNA (GFP on a plasmid)
Materials for each group (students should work in groups of 4):
Tube of plasmid DNA (tube labeled DNA)
One tube of E. coli bacteria (tubes on ice)
1 LB agar plate
2 LB agar plates with selection (ampicillin) (1 and 2 black stripes)
Q-tips or inoculation loops
1 tube of LB broth
micropipette
micropipette tips
small transfer pipette
large transfer pipette
Materials to share:
Water bath at 42°C
Ice
Trash containers/biohazard waste bag
UV lights
Protocol:
1. Pipette the 50 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?
3. In the meantime, each group should get one LB agar plate, two LB agar + ampicillin plate (one with one stripe
one with two stripes). Write your initials and your section number on your plates. You will be plating bacteria
with DNA on all three 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 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.
What is the LB broth for?
7. Add100 (first notch on the small transfer pipet), pipet 100 ml from your tube onto your LB agar plate and 100
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 stripe, and last LB agar + ampicillin plate with two stripes. --Why do you think you want to
spread the solutions onto the plates in this order?). Be careful not to stab the agar.
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?
1
BIOTECH Project, University of Arizona
Bacterial Transformation
The piece of DNA you will amplifiy is called the Green Fluorescent Protein (GFP) gene. This gene
codes for the GFP protein, a protein normally produced by jellyfish that you transformed into bacteria
in a plasmid (pGLO). The protein can be excited by UV light. In one group of plates (LB agar +
ampicillian) the bacteria transformed with the pGLO plasmid "glowed". In the other group of plates
(LB agar + ampicillian) the bacteria did not glow. Why do you think the bacteria glowed or did not
glow?
Extracting the DNA
Materials/Equipment Needed
For the class:
• Microwave
• Micropipet
• Micropipet tips
For each bacterial strain:
• Toothpick
• Sterile water
• 1.5 ml microfuge tube
• Bacteria colonies from both LB agar + ampicillian
Procedure
1. Pipet 20 µl sterile water into the microfuge tube.
2. Use a sterile pipet tip on a pipet to place a small amount of bacteria into the sterile water in the tube,
pipet the water/bacteria mixture up and down a few times to completely dislodge the cells.
3. Close the tube and microwave for 2 minutes on high to burst the cells and release their DNA.
4. The bacteria DNA is now ready for PCR. Store in a freezer until ready to use.
Conducting PCR
Materials/Equipment Needed
For the class:
• Thermocycler
• Microcentrifuge (optional)
• Micropipet
• Micropipet tips
For each reaction add the following to a PCR thin walled tube:
• 5 µl extracted DNA
• 10 µl 2.5X MasterMix (contains Taq polymerase, dNTPs, MgCl2, and buffer for ideal reaction.
• 10 µl primer mix (includes both the forward and reverse primers)
2
BIOTECH Project, University of Arizona
Bacterial Transformation
These are small volumes; you will need to be sure to look at the pipette tip when you are pipetting to
make certain that the components are being added.
Procedure
1. Label the microfuge tube so that you can distinguish DNA from the Amp plates with glowing
bacteria, or DNA from the Amp plates with nonglowing bacteria.
2. Using the micropipet with a clean tip, add 5 µl extracted DNA to the appropriate tube.
3. After adding the DNA, add 10µl master mix to the tubes. If necessary, gently tap your tube on the
counter to get all of the liquid to the bottom of the tube.
4. Add 10 µl of the primer mix to each tube. Close the tubes and centrifuge briefly (10 seconds) to
pool all of the liquid at the bottom of the tube.
5. Place your tube into the thermocycler to run the 'GFP' program. This program is 30 cycles of:
• 94°C for 30 seconds
• 55°C for 45 seconds
• 72°C for 1.5 minutes
6. After the cycles are complete, PCR reactions can be refrigerated or prepared for electrophoresis.
Electrophoresis of your PCR reactions
Materials/Equipment Needed
• Electrophoresis apparatuses, electrodes, and power supplies
• Micropipet
• Micropipet tips
• Loading dye
• 0.8% agarose gel
• Molecular weight markers (1 tube per gel)
• Water bath at 55°C or hot plate
• Thermometer for water bath
• TAE buffer
• Ethidium bromide paper (1 piece per gel)
• Staining tray
• Gloves (for handling ethidium bromide)
• UV light box
• UV light Polaroid set up (including camera, film, camera connector, and light shield)
• Biohazard bag
3
BIOTECH Project, University of Arizona
Bacterial Transformation
Procedure
Pouring an agarose gel
1. Get your electrophoresis apparatus and seal both ends of the gel tray with tape or stoppers.
2. Make sure one comb is in place at the negative electrode (black end of the gel).
3. Pour melted agarose into the gel space until the gel is about 5 mm deep. Let the agarose
harden, which should take 5-10 minutes. Don’t touch/move your gel until it’s hard. In the
meantime, prepare your PCR reactions for electrophoresis.
Electrophoresis of your PCR reactions
1. Using the micropipet with a clean tip, pipet 5 µl gel loading dye into your PCR reaction tube.
You will load both your PCR reactions and standard DNA markers sample into the gel. A standard
DNA marker has a bunch of different sized pieces of DNA so you can compare it to the DNA from
your PCR reaction to figure out what size piece it is. Two or three groups can share a gel, but only one
molecular weight marker is needed per gel.
2. Draw a picture of your gel and label in which wells you will load which samples (PCR reaction(s),
DNA marker). Be certain to have the information of where the other groups added their samples.
3. When your gel has hardened, remove the tape or stoppers.
4. Load your samples into the wells - be sure you keep track of which samples you're loading in which
wells.
5. Pour TAE buffer carefully so it fills the electrophoresis apparatus and just covers the gel.
6. Run that gel! Plug the electrodes into your electrophoresis apparatus (red to red, black to black),
being careful not to bump your gel too much.
7. Plug the power source into an outlet and set the voltage to about 100 V (max = 120 V).
8. Let the gel run until the dye migrates about 5-6 cm from the wells (about 20-25 minutes).
9. Turn off the power supply, disconnect the electrodes, and remove the top of the electrophoresis
apparatus.
10. Carefully remove the gel. The gel can be wrapped in plastic wrap and stored in the refrigerator or
placed it in the staining tray for DNA staining.
4
BIOTECH Project, University of Arizona
Bacterial Transformation
Staining gels to examine PCR reactions
1. Place gel in staining tray
2. Using gloves, remove the plastic from the ethidium bromide sheet and place the ethidium bromide
paper on the gel. Gently rub the paper with your fingers to make sure it is contacting the gel all
over.
3. Stain for about 10 minutes.
4. Put the gel on the UV light box and, with the UV shield down, view your gel.
5. Take a Polaroid picture of your gel.
Analysis
• What do you see on your gel? Which bacteria samples were able to amplify the GFP gene from the
pGLO plasmid? Why do you think you saw these results? What else could you do to ascertain the
amplified DNA is the GFP gene?
This product was funded by a grant awarded under the President’s High Growth Job Training Initiative as implemented by the U.S.
Department of Labor’s Employment & Training Administration. The information contained in this product was created by a grantee
organization and does not necessarily reflect the official position of the U.S. Department of Labor. All references to non-governmental
companies or organizations, their services, products, or resources are offered for informational purposes and should not be construed as
an endorsement by the Department of Labor. This product is copyrighted by the institution that created it and is intended for individual
organizational, non-commercial use only.
5