Determining Species of Bacteria
In this lab investigation, you will use a piece of DNA to identify bacteria. The piece of DNA
used is the region that codes for a small subunit of the ribosomal RNA (16S rRNA). Different
bacterial species have unique 16S rDNA sequences. The identification relies on matching the
sequence from your sample against a database of all known 16S rDNA sequences.
• In order to do this you will make many copies of the 16S rDNA. How can you make copies of
DNA? What does your cell use to make copies of its DNA?
• Make a list of steps involved in copying DNA:
You will use some of these same ingredients and steps to copy DNA in a test tube instead of a
cell. Compare the ingredients and steps use to copy DNA in the test tube to that of the cell.
Conducting PCR
Materials/Equipment Needed
For the class:
• Thermocycler
• Microcentrifuge (optional)
• Micropipet and sterile tips
• Bacteria colonies
For each reaction add the following to a PCR thin walled tube:
• 15 µl 2X GoTaq (contains Taq polymerase, dNTPs, MgCl2, and buffer for ideal reaction and
loading dye for electrophoresis).
• 7.5 µl forward primer
• 7.5 µl reverse primer
•
bacteria from one colony
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.
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Procedure
1. Label the PCR tube so that you can distinguish the samples in the tube.
2. Add 7.5 µl primer of each primer to each tube. If necessary, gently tap you tube on the counter
to get
all of the liquid to the bottom of the tube.
3. Add 15 µl GoTaq (green solution). Close the tubes and centrifuge briefly (10 seconds) to
pool all of the liquid at the bottom of the tube (if you do not have a centrifuge then tap or
shake the tube contents to the bottom of the tube).
4. Using the micropipet with a clean tip, scrape one of the colonies that you would like to
amplify the 16S rDNA. You will need a fair amount of the colony, so you should see the
bacteria on the tip. Place the tip into the PCR tube and pipet up and down once or twice to
dislodge the bacteria and mix.
5. Place your tube into the thermocycler to run the '16S' program. This program is:
• 94°C for 5 minutes
30 cycles of
• 94°C for 30 seconds
• 49°C for 45 seconds
• 72°C for 1 minute
Final extension
• 72°C for 7 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
• 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
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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. You will load 15 µl of your PCR reactions and 10 µl of a 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. Be sure to
save your remaining PCR sample to send off to sequencing.
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. Be sure to save your remaining PCR sample to send off to sequencing.
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.
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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 picture of your gel. Draw a picture of your gel:
Analysis
What do you see on your gel? Which bacteria samples were able to amplify 16S rDNA? What
was the size of the PCR product? You will send this sample to be sequenced and compare the
sequence to known pieces of 16S rDNA sequences to determine what your sequence is most like.
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