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Name __________________ Period _____ Date _________
Lesson 3: Soil Microbes, What are they doing?
Purpose:
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To learn how to set up a Polymerase Chain Reaction
(PCR) for bacterial amoA and bacterial 16S genes.
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To determine if nitrifying bacteria are also
involved in ammonia oxidation.
SAFETY:
Follow ALL the instructions to make sure that:
 None of the organisms you grow escape into the environment
 You do not pick up any infections from the material you touch.
Day 1 – Setting up PCR
Procedure (check off boxes at each step!)
Materials:
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PCR tubes and lids
20 µl pipette and tips
20-200 µl pipette and tips
Optional: 10 µl pipette and tips
2 - 1.5 ml Eppendorf tubes
Master Mix for 16S and amoA reactions
200 µl PCR water (nuclease-free)
Autoclaved toothpicks or inoculating loop with alcohol lamps
Bacteria plates with bacterial growth from Lesson 2 – Nitrogen-free
medium and nutrient medium
Pens for labeling
Note: Use sterile technique
1.  Wear gloves at all times when working with bacteria!
2.  Wipe down your work area with alcohol.
3.  Label PCR tubes according to your teacher’s instructions
VALERY LYNN, IISME 2011
Name __________________ Period _____ Date _________
4.  Transfer 100 µl PCR water to 2 Eppendorf tubes labelled “Nitrogen free”
and “nutrient.”
5.  Using sterile technique, transfer a toothpick or inoculating loopful from
each colony into the PCR water tubes. Shake or vortex vigorously! These
are your Bacterial Mixes
6.  Transfer 24 µl of Master Mix in each PCR tube according to your
teacher’s directions.
7.  Transfer 1 µl from each of your Bacterial Mixes into your reaction PCR
tubes according to your teacher’s directions.
8.  Don’t put anything other than the Master Mix into your negative control
tube.
9.  Give your tubes to your teacher to load in the thermocycler.
10. In your lab notebook, write down a brief description of your methods (what
you just did.)
Question: What results do you expect from the 16S PCR? The amoA PCR?
Write down in your notebook.
Day 2 – load and run agarose gels to view results of the PCR.
Procedure
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Loading dye
DNA Ladder Mix (0.1 µg/µl) - GeneRuler™
10 or 20 µl pipette and tips
Parafilm or wax paper
1. Cut a small piece (about 2 X 4 in) of parafilm or wax paper.
2. Load 5 µl of loading dye into your pipette and tip.
3. Drop 4 drops of loading dye spaced evenly across the parafilm or wax
paper. Each drop will be about 1 µl. You will have a little left in the tip when
you are done.
VALERY LYNN, IISME 2011
Name __________________ Period _____ Date _________
4. Now transfer 5 µl from each of your PCR tubes and mix with each drop of
dye (pipette up and down to mix).
5. Set your pipette to 6 µl. When it is your turn to load the gel, do the
following:
a. Load the DNA ladder (6 µl) in well #1
b. Now load each of your mixed drops from the parafilm/wax paper
into the next 4 wells. Make sure to keep them in order!
c. Load the DNA ladder (6 µl) in well #6
6. Draw pictures in your lab notebook of what you think your gels will look like
after they run! Think about the questions you answered on Day 1. What
results do you expect?
Conclusions
1. Did your results support your predictions? What does this mean in terms of
the genes your bacteria have? Write your conclusion as a paragraph in your
lab notebook. Include the need for further studies, and how they should be
conducted if your results did not support your hypothesis.
Questions
1. What is the significance of the 16S ribosomal gene?
2. What is the significance of the amoA gene?
3. Draw the nitrogen cycle and discuss how enzymes are involved.
4. Are the same bacteria that are nitrifying (taking nitrogen out of the
atmosphere) also involved in ammonia oxidation? How can you tell?
VALERY LYNN, IISME 2011
Name __________________ Period _____ Date _________
Answers
1. What is the significance of the 16S ribosomal gene? This gene is
present in all bacteria. It is a way to determine that the
colonies tested were actually bacteria, and not something else.
2. What is the significance of the amoA gene? This gene codes for
one of the enzymes that converts ammonium to nitrate – an
important step in the nitrogen cycle
3. Draw the nitrogen cycle and discuss how enzymes are involved.
Students should reproduce the nitrogen cycle and discuss how
enzymes catalyze reactions.
4. Are the same bacteria that are nitrifying (taking nitrogen out of the
atmosphere) also involved in ammonia oxidation? How can you tell?
No, these two steps are done by different bacteria. Students
should have gotten positive results for bacteria living on a
nitrogen-free medium, but negative results for the PCR of amoA.
This suggests that nitrifying bacteria are not involved in ammonia
oxidation.
VALERY LYNN, IISME 2011