Elizabeth Blosky
Bacteria Primer Comparison PCR Lesson Plan
Goal: The goal of this lesson is for students to hypothesize which primer they think will work best when
used on the same three types of bacteria. This lesson should be done after students have read about
and discussed the process of PCR and primers as well as after they have practiced PCR through a virtual
simulation. This lesson will be followed by a gel electrophoresis lesson for students to compare the
primer results. Students will also have the chance to familiarize themselves with the online database
PubMed to research types of bacterial characteristics.
Keystone Objective:
Students will be able to explain how genetic engineering has impacted the fields of medicine, forensics,
and agriculture (e.g., selective breeding, gene splicing, cloning, genetically modified organisms, gene
therapy, gel electrophoresis, PCR).
Lesson Procedure:
1) Order at least 3 bacterial samples for comparison purposes. (Pseudomonas aeruginosa,
Escherichia coli and Corynebacterium pseudodiptheriticum are recommended since they are all
rod shaped, can be found in the human body and can be ordered from the same supply
company-Wards Science Supply Company).
2) Order two primers for students to compare which one is more effective at amplifying the
intergenic space between the 16S gene and the 23S gene in bacteria. Both of these genes code
for rRNA. The two suggested primers to us are: Card_ITSF/Card_ITSReub and
SDBact1522bS20/LDBact132aA18.
a. Both of these primers can be ordered through IDT (integrated DNA technologies).
b. These primers need to be diluted. (example: 90uL of water to 10uL of primer)
3) Culture the bacteria to grow on agar plates to obtain samples of each type of bacteria.
4) Before starting the PCR protocol, students should hypothesize which primer they think will
amplify the intergenic space between the 16S gene and the 23S gene in bacteria. This could be
a hypothesis made without any previous knowledge about the primers and their relationships
with prokaryotes or students could be assigned to research the primers before starting this
lesson by using a search engine such as Google to locate ARISA.
5) Follow the PCR protocol for bacteria which is intended to amplify the intergenic space between
the 16S gene and the 23S gene in bacteria.
a. Create a Master mix:
i. 20 uL total for each PCR tube
ii. 2uL of PCR buffer
iii. .8uL of MgCl2
iv. .4uL of dNTP
v. .4uL of forward primer
vi. .4uL of reverse primer
vii. .2uL of Taq polymerase
viii. 15.8uL of water
ix. Note: The above amounts are for a master mix in ONE PCR tube. Since this
lesson utilizes three bacterial cultures, multiply 3 by the numbers above to
make one larger master mix. It was recommended not to make a master mix
more than 20X.
b. PCR Instructions:
i. put 20 uL of master mix into each PCR tube
ii. using a sterile toothpick, inoculating loop, or pipette tip, take one colony off the
plate and stir in PCR tube (with master mix)
iii. repeat step 2 for each PCR tube.
iv. Run PCR with the following protocol
1. 94 degrees Celcius – 5 minutes
2. Cycle: 35 times
3. 94 degrees Celcius – 30 seconds
4. 53 degrees Celcius – 30 seconds
5. 72 degrees Celcius- 1 minute
6. Final: 72 degrees Celcius – 5 minutes
6) After preparing the master and starting the PCR, students should research the three types of
bacteria used during this lab by searching and reading related articles on PubMed
(http://www.ncbi.nlm.nih.gov/pubmed/). Students should identify and describe where each
type of bacteria can typically be located, if it is motile, its shape and if it is a gram positive or
gram negative reaction. (Since this class period is only 43 minutes, this research may need to be
conducted the day before this lesson. If it is a block schedule class period, it could be done while
waiting for the PCR reaction to be completed.)
Type of Bacteria
Where bacteria
can be located
Motile or Nonmotile
Bacterial Shape
Gram positive or
gram negative