miniPCR COVID QPCR Instructor Guide V1 1 032322

COVID-19 qPCR Lab
Detecting
SARS-CoV-2
Infection
For educational use only. Not for diagnostic use.
COVID-19 qPCR Lab Version: 1.1

Detecting SARS-CoV-2 Infection Release: April 2022
Instructor’s and Student’s Guide © 2022 by miniPCR bio™
Instructor’s Guide
Instructor’s
Guide Contents
Getting Started
At a glance P.03
Materials needed P.06
Lab setup P.09
Student’s Guide
Background information P.13
Today’s lab P.19
Laboratory guides P.21
Extension: Quantification with qPCR P.60
Additional background: COVID-19 testing P.62
Expected results P.65
Notes on lab design P.67
Differentiation P.67
Additional student supports P.68
Extension activities P.68
Placement in unit P.69
Learning goals and skills developed P.69
Standards alignment P.70
Ordering information P.71
About miniPCR bio Learning Labs™ P.72
miniPCR bioTM COVID-19 qPCR Lab: Detecting SARS-CoV-2 Infection - Instructor’s and Student’s
Guide Version: 1.1 - Release: April 2022 - © 2022 by miniPCR bio™ P./2
At a glance
Practice the gold standard technique used to test patients for COVID-19!
Students will use low-cost tools to get hands-on experience with qPCR principles and determine if
fictional patients are infected with the SARS-CoV-2 virus.
Disclaimer: no pathogenic materials are used. This experimental protocol engages students in a
simulated patient diagnosis exercise. None of the materials provided in this lab kit pose a pathogenic
risk. For educational use only. Not for diagnostic use.
TECHNIQUES TOPICS LEVEL WHAT YOU NEED AP CONNECTION
Micropipetting Infectious disease General high Micropipettes AP Biology units 6.8

Quantitative PCR Molecular school PCR thermocycler
Fluorescence diagnostics Advanced high P51™ molecular fluorescence Skills and Practices
visualization Biotechnology school viewer or other blue light 1.A-1.C, 2.A, 2.D,
Gel electrophoresis College illuminator 3.A-3.D, 4.A-B, 6.A-6.E
(optional) Optional: Gel electrophoresis
and visualization system
Planning your time

-
The data for this lab can be collected two ways: endpoint observation or qPCR time point
observations. See pages 4-5 for details.
Option 1: Endpoint observation Option 2: qPCR time point observations
SINGLE CLASS PERIOD: 50 MINUTES SINGLE CLASS PERIOD: 80 MINUTES
OR
PERIOD 1: PERIOD 2:
35 MINUTES 5 MINUTES
Help your students build proficiency in pipetting, PCR, and gel electrophoresis with
Additional additional instructional videos, worksheets, and activities available at:
supports
https://www.minipcr.com/tutorials/
-
Visit https://www.minipcr.com/covid-resources/ to access the complete miniPCR
bio™ COVID-19 resource library.
For answers to the lab study questions and extensions, email answers@minipcr.com. Please include the name of the lab, as
well as your name, school, and title in the body of the email.
Option 1: Endpoint observation

For classes with shorter class periods or where an emphasis on the quantitative aspects of qPCR is
unnecessary, we recommend students collect a single endpoint observation (instructions on pages
22-27). This approach takes less class time and still allows students to diagnose their patients as
being infected or uninfected. However, it does not allow students to compare the relative viral loads
between samples. This approach can be completed in a single 50 minute class period, or split
across two class periods. Further, this method of data collection can completed using a single
thermal cycler that is shared across lab groups if the machine is large enough. For eight lab groups,
you will have 48 samples..
SINGLE CLASS PERIOD: 50 MINUTES

OPTIONAL
OR 30 MINUTES*
PERIOD 1: 35 MINUTES PERIOD 2: 5 MINUTES
Possible
stopping
point
Once PCR has started, samples may be left unattended.

PCR products are stable at room temperature for at least
one week. See note below for instructions to collect preliminary
observations before the PCR program is complete.
Prepare View and Optional: Gel

Run PCR
PCR samples interpret results electrophoresis
Primer
DNA
* The PCR program will * Students will perform a * Allow an additional 25

take ~35 minutes. single endpoint observation minutes if students will be
of fluorescence. preparing the gels in class.
Note: If students run out of class time before their PCR is done, they can pause the PCR program
during the extension step (72°C) and collect initial observations. Students can then return their
samples to the thermal cycler for the PCR program to finish and observe their final results the next
class. See page 65 for details.
Option 2: qPCR time point observations

For classes where it is important to teach about quantitative PCR in detail, we also include
instructions for students to collect data throughout the PCR (pages 37-44). This approach allows
students to generate qPCR curves and compare viral loads across samples.
This option requires:
• A single 80 minute class period as students need to both set up the PCR and then monitor
the fluorescence throughout the PCR program.
• Thermal cyclers must have the capability to pause a PCR program while it is running (e.g.
miniPCR®)
• Each student group will also require their own blue light illuminator to make frequent
observations (e.g., P51™ molecular fluorescence viewer or blueGel™).
OPTIONAL
SINGLE CLASS PERIOD: 80 MINUTES 30 MINUTES*
Run PCR and collect several

Prepare Optional: Gel
fluorescence measurements
PCR samples electrophoresis
over time
Primer
DNA
* The PCR program will take * Students will perform * Allow an additional 25
~50 minutes to complete when repeated observations minutes if students will be
students collect time point of fluorescence. preparing the gels in class.
observations.
Materials needed
Supplied in kit (KT-1900-06)
Amount Amount
Reagents provided needed per Teacher’s
and supplies in kit lab group Storage checklist
2X qGRN Master Mix 1000 μl 110 μl Freezer
COVID Lab Primers 550 μl 55 μl Freezer
Simulated Patient Samples 100 μl each 10 μl each Freezer

• Patient AH
• Patient BH
• Patient CH
• Patient DH
Simulated Control Samples 100 μl each 10 μl each Freezer

• Positive Control
• Negative Control
PCR strip tubes Ten 8-tube One 8-tube Room

strips strip temp.
For optional gel electrophoresis:
6X Loading Dye 300 μl 25 μl Freezer
Fast DNA Ladder 1 150 μl 15 μl Freezer
Materials needed (cont.)

Supplied by teacher
Reagents Teacher’s
and supplies Amount needed per lab group checklist
PCR thermal cycler: e.g. miniPCR® machine 1
Note: If students are collecting time point qPCR Six reactions per group
observations, the machine must have the Can be shared between groups
capability to pause while running a PCR if performing endpoint
program observation
P51™ molecular fluorescence viewer 1

Available at miniPCR.com
or other blue light illuminator: Can be shared between groups

e.g. blueGel™ or blueBox™ if performing endpoint observation
Micropipettes
• 2-20 μl: one per lab group 1
• 20-200 μl: one for the teacher to dispense
reagents
Disposable micropipette tips At least 18 per group
PCR tubes (0.2 ml): Included in the Lab 6

Companion Kit (see next page for details)
Plastic tubes (1.7 ml): Included in the Lab 8

Companion Kit (see next page for details)
Other supplies:
• Disposable laboratory gloves
• Protective eyewear
• Permanent marker
• Cup to dispose of tips
Additional materials needed for

optional gel electrophoresis
Supplied by teacher
Reagents Amount Teacher’s
and supplies needed checklist
Horizontal gel electrophoresis apparatus 1 per group
e.g. blueGel™ electrophoresis system Can be shared between groups if you use
two combs per gel
Distilled water for making agarose gels and 50 ml per gel

diluting TBE buffer
Heat-proof flask or beaker to dissolve 1 per class

agarose
Microwave or hot plate to dissolve agarose 1 per class
Sold separately in Learning Lab Companion Kit

(KT-1510-01)
This lab requires reagents for running and visualizing DNA samples on a 2% agarose gel with a
fluorescent DNA stain (e.g., SeeGreenTM or GelGreen®). The Learning Lab Companion Kit provides
enough electrophoresis reagents for 8 groups when using the blueGel™ electrophoresis system. Gels
can also be prepared with agarose tabs or agarose powder. Refer to www.minipcr.com/agarose-gel/
for detailed instructions..
Amount Amount
Reagents provided needed per lab Teacher’s
and supplies in kit group Storage checklist
All-in-one agarose tabs 8 One tab per Room temp.,
with DNA stain and TBE agarose gel protected
(2% agarose gel) from light
TBE electrophoresis Supplied as liquid 30 ml 1X solution Room temp.

buffer concentrate or per blueGel™
powder system
Sufficient for
600 ml of 1X
working solution
PCR tubes (0.2 ml) 100
Plastic tubes (1.7 ml) 50
Lab setup: PCR and fluorescence

visualization
Note: The reagents setup for both endpoint observation and qPCR time point observations is the
same.
The following activities can be carried out by the instructor ahead of class. Reagents are sufficient
to be used with 8 student groups. Reagents are stable at room temperature for 24 hours but should
remain cold for short-term storage and frozen for long-term storage.
Gloves and protective eyewear should be worn for the entirety of this lab.
Preparation for PCR Defrost tubes
• Thaw tubes containing 2X qGRN Master

Mix, COVID Lab Primers, Simulated Patient
Positive Control
Negative Control
Master Mix
Patient AH
Patient CH
Primers
Patient BH
Patient DH
Samples, and Simulated Control Samples by
placing them on a rack or benchtop at room
temperature.
Aliquot reagents
• For each lab group, label and dispense the
following reagents into eight labeled 1.7 ml
MM
tubes:
P
- 2X qGRN Master Mix 110 μl

- COVID Lab Primers 55 μl
- Simulated Patient Samples 10 μl each 110 μl 55 μl
Master Primers
• Patient AH Mix
• Patient BH
• Patient CH
B
A
• Patient DH
number
- Simulated Control Samples 10 μl each of groups
• Positive Control up to 8
10 μl 10 μl 10 μl 10 μl
• Negative Control Patient AH Patient BH Patient CH Patient DH
• Distribute supplies and reagents to lab

Pos
Neg
groups (continued on next page).
10 μl 10 μl
Positive Negative
Control Control
Check At the start of this experiment, every lab group should have: Amount
2X qGRN Master Mix 110 μl
COVID Lab Primers 55 µl

Simulated Patient Samples 10 μl each
- Patient AH
- Patient BH
- Patient CH
- Patient DH
Simulated Control Samples 10 μl each
- Positive Control
- Negative Control
PCR tubes (200 μl) 1 strip of 8 tubes
2-20 μl micropipette 1
Micropipette tips At least 18
Fine tipped permanent marker 1
6 wells in a miniPCR® or other thermocycler

Note: If students are collecting time point qPCR
observations, the machine must have the
capability to pause while running a PCR
program
Access to a P51™ or other blue light illuminator
Hardware setup
• Thermal cyclers can be programmed ahead of time

by the teacher or during class by the students.
• If doing option 1 (endpoint observation):
° If your thermal cycler is large enough, a single
machine can be shared across groups. For
eight lab groups, you will have 48 samples.
° A single, shared blue light illuminator is
sufficient, although one per lab group will
provide a better student experience.
• If doing option 2 (qPCR time point observations):
° One thermal cycler per lab group is required.
° Thermal cyclers must have the capacity to
pause during run (e.g., miniPCR® app).
° One blue light illuminator per lab group is
required (e.g., P51™ fluorescence viewer).
Lab setup: Optional gel

electrophoresis
We recommend examining the PCR products with gel electrophoresis, but this can be skipped in the
interest of time.
Preparation for gel electrophoresis

• For each lab group, label and dispense the following
reagents into two labeled 1.7 ml tubes:
Dye
L
- 6X Loading Dye 25 μl
- Fast DNA Ladder 1 15 μl
• Have the banding pattern of the Fast DNA Ladder 1 handy
(page 57) to help interpret the electrophoresis results. 25 μl 15 μl
Loading Dye Ladder
• Distribute supplies and reagents to lab groups:
Check At the start of this experiment, every lab group should have: Amount
PCR products from previous section of the lab 6 reactions
6X Loading Dye 25 µl
Fast DNA Ladder 1 15 μl
2-20 µl micropipette 1
Micropipette tips At least 13
7 wells in an electrophoresis gel
Preparing gels
• Prepare 1X TBE buffer.
- TBE buffer is often provided as liquid concentrate or powder.
- Follow manufacturer’s instructions to prepare 1X TBE buffer solution.
- Volume to prepare depends on the method used to prepare gels; see “Important
Note” below.
• Gels can be poured in advance of the class.
- This lab requires running and visualizing DNA samples on a 2% agarose gel with a
fluorescent DNA stain (e.g., SeeGreenTM or GelGreen®).
- Pre-poured gels can be stored at ambient temperature, in a sealed container or
wrapped in plastic wrap, and protected from light for up to three days.
IMPORTANT NOTE: There are several ways to prepare agarose gels.
• Scan the QR code for detailed instructions on how to prepare

agarose gels.
• Both written and video instructions are available.
www.minipcr.com/agarose-gel/
Student’s Guide
Student’s
Guide Contents
Background information P.13
Today’s lab P.19
Laboratory guide 1: Endpoint observation P.22
Laboratory guide 2: qPCR time point observations P.37
Laboratory guide 3: Optional gel electrophoresis P.53
Extension: Quantification with qPCR P.60
Additional background: COVID-19 testing P.62
miniPCR bioTM COVID-19 qPCR Lab: Detecting SARS-CoV-2 Infection - Instructor’s and Student’s Guide
Version: 1.0 - Release: January 2022 - © 2022 by miniPCR bio™ P./12
Student’s Guide
Background Important acronyms

• SARS-CoV-2: severe acute respiratory syndrome
information coronavirus 2. A novel virus that emerged in

human populations in 2019.
• COVID-19: coronavirus disease 2019, the disease
resulting from infection with SARS-CoV-2 virus.
As a healthcare provider, your job is to diagnose your
Membrane envelope
patients and provide the best treatment possible
based on that diagnosis. However, it can be difficult Protein coat Surface
proteins
to tell different illnesses apart because symptoms of
many diseases are similar.
RNA genome
Today, you will test four travelers for COVID-19.
As their healthcare provider, you’ll use molecular
techniques to determine whether your patients are
infected with SARS-CoV-2 (pronounced “sars-co-
vee-two”), the virus that causes the disease known as
COVID-19.
SARS-CoV-2 Figure 1. SARS-CoV-2 structure. The SARS-CoV-2

virus has an RNA genome packaged inside a protein
- coat and a membrane envelope. There are a handful
of viral membrane surface proteins, including the
spike protein. The spike protein binds with receptors
Viruses are infectious agents with relatively simple
on host cells to gain entry and begin the viral
structures: all viruses contain a small genome made replication cycle.
of RNA or DNA surrounded by a coat made of protein
1
and sometimes lipids (Figure 1). Because they must Host cell receptor
use a host’s cellular machinery to reproduce, viruses Host

are generally considered nonliving. SARS-CoV-2 virus cell
2
A virus must invade a living cell and use the cell’s 4
machinery to make copies of itself (Figure 2). Viruses

3
recognize molecules on the outside of the host cell
and then enter the cell. The SARS-CoV-2 virus is
covered in specialized proteins called spike proteins
that recognize and bind to specific receptors on
human (and other mammalian) cells. This interaction
between the virus and the host cell receptor triggers Figure 2. SARS-CoV-2 viral replication cycle.
(1) The SARS-CoV-2 spike protein binds to host cell
the entry of the virus into the cell. receptors and enters the cell. (2) The virus particle
sheds its coat, releasing its RNA genome into the
host cell. (3) The host cell copies the viral genome
and expresses viral proteins, which come together to
make new virus particles. (4) Newly assembled virus
particles emerge from cell, ready to infect new host
cells.
Student’s Guide
Once inside the cell, the viral genome is released from the protein coat, and the molecular machinery
within the host cell manufactures viral proteins and replicates the viral genetic material. Newly made
viral proteins and copies of the viral genome then come together to form new virus particles. These
viruses emerge from the host cell, moving on to infect new cells—and new hosts if they’re able to
find their way out of the body, say through a cough or sneeze—and the cycle repeats itself.
While an infection can be disastrous for the host, the speed and efficiency of viral replication
represent a success from the virus’s point of view. Despite their small size, simple construction, and
total dependence on living cells, viruses are remarkably efficient in achieving the primary functions
of all biological entities: to persist and replicate.
Diagnosing SARS-CoV-2 infection

-
Different respiratory viruses that infect our airways often produce very similar symptoms, making
it difficult to tell which virus underlies a person’s particular illness. For example, a cold and a case
of seasonal influenza may appear quite similar, both causing a cough, nasal congestion, and body
aches. However, these diseases are caused by different viruses, and correctly diagnosing the
infection can be important to treat the patient properly.
For COVID-19, quickly determining whether a person’s symptoms are due to SARS-CoV-2 is essential
for properly monitoring a person’s disease and preventing further spread of the virus. While doctors
use numerous lab tests to diagnose SARS-CoV-2 infection1, we will focus on nucleic acid detection
tests today.
Nucleic acid tests are used to diagnose many conditions, including both viral and bacterial
infections. The key principle is that unique genetic sequences can be used to identify pathogens. For
example, if genetic sequences that are unique to the SARS-CoV-2 virus are detected in a patient’s
sample, their doctor can diagnose them as being infected with that specific virus.
1
For information comparing different COVID tests, refer to the Additional background, page 62.
Student’s Guide
A SARS-CoV-2 nucleic acid detection test typically involves the following three steps (Figure 3):
Step 1 - Collect patient sample:

For a respiratory virus like SARS-CoV-2, the nasal passage is typically swabbed to collect viral
particles.
Step 2 - Extract genetic material:

The cells and virus particles are ruptured to release their DNA and RNA. Because SARS-CoV-2 is an
RNA virus, scientists specifically isolate RNA from the sample. This will include any RNA present on
the swab, and will likely include RNA from the patient and RNA from any bacteria or viruses present
in the nasal passage. The detection of RNA viruses like SARS-CoV-2 requires an intermediate step
where RNA is converted into DNA in a process called reverse transcription. Once the RNA has been
converted into DNA, scientists attempt to copy one or more gene segments specific to the SARS-
CoV-2 virus.
Step 3 - Amplify and analyze viral sequence:

Scientists commonly use a technique called polymerase chain reaction (PCR) to copy SARS-CoV-2
genetic sequences. If the PCR copies the gene, it means that the SARS-CoV-2 virus was present in
the sample. If the PCR does not generate copies of the gene, it means that the SARS-CoV-2 virus
was not present.
Bacteria
RT
Other
viruses
Human RNA isolated & Amplification of specific
cells SARS-CoV-2 copied into DNA SARS-CoV-2 gene
Extract and process Amplify and visualize

Collect sample
genetic material viral gene
Already completed You will complete this step
Figure 3. SARS-CoV-2 nucleic acid detection. (1) A sample is collected from the patient, often by swabbing the nasal
passage. This sample will contain cells from the patient, as well as any bacteria and viruses that are present. (2) Because
SARS-CoV-2 has an RNA genome, scientists isolate RNA from the patient sample. Next, reverse transcription is used to
convert the RNA to DNA. This is necessary because the final step, PCR, only works on DNA. (3) To detect specific SARS-
CoV-2 genetic sequences, PCR is used. PCR copies specific genetic sequences, allowing scientists to determine if the
SARS-CoV-2 virus was present. Finally, scientists analyze the result of the PCR to see if the SARS-CoV-2 viral genetic
sequence was detected.
Student’s Guide
Quantitative PCR (qPCR)

- 4th Cycle
Target DNA
PCR allows scientists to copy a specific
sequence of DNA, known as the target 3rd Cycle
sequence. During PCR, the number of

copies of the target DNA sequence 2nd Cycle
doubles with each cycle, leading to
exponential amplification (Figure 4).
Theoretically
unlimited
The ability to copy a specific genetic 1 Cycle
st
exponential
sequence—and only that sequence—is expansion
essential in a COVID test because the

sample will contain a complex mix of
genetic material from the patient’s
21 =
own cells as well as any bacteria or 2 copies
viruses that were present. In a COVID

22 =
test, scientists use PCR to copy a gene 4 copies
8 copies
specific to the SARS-CoV-2 virus. If 16 copies
the PCR copies the viral gene, it tells

Figure 4. Exponential amplification using PCR. During PCR, the
you that the SARS-CoV-2 virus was
number of copies of the target DNA doubles with each cycle. At the
present, and the patient is diagnosed end of a typical PCR run, billions of copies of the specific target have
as being infected.2 been generated. While the input for PCR during a COVID test is a
complex DNA sample with patient DNA, as well genetic material from
any bacteria or viruses that were present, PCR allows scientists to copy
Scientists commonly use a variation just the genetic sequence they are interested in. In a COVID PCR test,
of PCR called quantitative polymerase scientists amplify a SARS-CoV-2 specific gene. If the PCR copies the
viral gene, it tells you that the SARS-Cov-2 virus was present, and the
chain reaction (qPCR) to diagnose patient is diagnosed as being infected.
SARS-CoV-2 infection. qPCR copies
the DNA and uses a fluorescent readout to simultaneously detect whether or not copies were made.
qPCR is able to track how much of the target sequence is produced in each PCR cycle using
a fluorescent dye or probe. This can be done a few different ways, but the simplest is to add a
fluorescent dye that will only bind to double-stranded DNA. As the qPCR progresses and makes
more and more copies of the target sequence, the tube will fluoresce brighter and brighter. How
brightly the reaction fluoresces is a direct readout of how much DNA is in the tube. For some
viruses, estimating the amount of viral genetic materiel present in a patient sample, or viral load,
can help inform treatment.
When qPCR is performed in a research lab or medical facility, fluorescence is monitored throughout
the qPCR by an automated fluorometer—people never actually look at the samples. The qPCR
machine produces a graph where the relative fluorescence of each sample is plotted over time
(Figure 5), and scientists use these graphs to interpret the qPCR results.
2
A typical COVID-19 PCR assay tests for two SARS-CoV-2 genes at the same time.
Student’s Guide
Relative fluorescence
Sample 1
Sample 2
Sample 3
PCR cycle number
Figure 5. qPCR monitors PCR products over time using fluorescence. During qPCR, a fluorescent dye or probe is used
to track the accumulation of PCR products. As copies of the target DNA double with each PCR cycle, fluorescence levels
increase and eventually cross a threshold where they are detectable. The more copies of the target sequence that are
present in the sample at the start of the PCR, the sooner the threshold will be crossed. In the graph above, Sample 2
had the most copies of the target sequence since the fluorescence for Sample 2 became observable the soonest, after 11
cycles (blue arrowhead). On the other hand, the florescence in Sample 3 became detectable at cycle 14 (pink arrowhead).
Comparing two samples at the same time point allows relative quantification. For example, comparing Samples 2 and 3
at cycle 18 (dashed line) also shows that Sample 2 has more product than Sample 3. In this experiment, Sample 1 did not
contain the target sequence as evidenced by the lack of florescence.
While there are multiple options for diagnosing SARS-CoV-2 viral infections, qPCR is considered the
gold standard for several reasons. First, PCR-based assays are extremely sensitive and can detect
infections where relatively few copies of the virus are present. Second, qPCR can be streamlined and
automated to reduce the chance of human error or contamination. In many cases, the entire process
is automated and can take place in a single tube. The qPCR machine monitors the fluorescence
throughout the reaction, and algorithms are applied to determine whether each sample should be
categorized as infected or uninfected.
PCR vs. qPCR

PCR is one of the most common and powerful techniques in a molecular biology laboratory. PCR gives scientists the
ability to find a specific sequence of DNA in a complex sample and make billions of copies of that specific sequence.
But while PCR is an incredibly powerful tool, it is typically an endpoint reaction; this means that once we start a PCR,
we don’t look at it again until the reaction is over. Quantative PCR (qPCR) puts a spin on this process by monitoring the
reaction in real time, using fluorescence to label the copies of DNA as they are produced. During qPCR the amount of
fluorescence that is measured is proportional to the amount of DNA being produced—the brighter it glows, the more
DNA is present. By tracking the rate of the reaction and comparing that rate to other reactions, it is pos sible to calculate
relatively how much of the very specific DNA sequence you started with. For more information, refer to the extension on
page 60.
Its ability to not only detect, but also quantify, a genetic sequence of interest has made qPCR a powerful tool in medical
diagnostics. By using qPCR, a physician can tell not only if a particular DNA sequence is present, but they can also tell
whether it is abundant or rare. For example, a physician may be interested in whether a virus is present in the body, as
well as the patient’s viral load. qPCR can answer both questions in a single test. Because of this, qPCR is routinely used
to diagnose infections with SARS-CoV-2, HIV, hepatitis, and many other viral pathogens.
Student’s Guide
qPCR is a powerful tool for diagnosing viral infections when you have access to a qPCR machine in a
laboratory. However, sometimes it is necessary to deploy testing directly at the point of care. qPCR
machines are not well-suited for use outside of a laboratory because they are large (think the size of
a microwave or larger), delicate, and expensive, typically costing tens of thousands of dollars.
To address these issues, scientists have developed SARS-CoV-2 nucleic acid detection tests that
utilize the same principles as qPCR, but that can be performed without the use of a qPCR machine.
Instead of using a specialized qPCR machine, you can use a standard PCR machine and remove the
samples to judge the amount of fluorescence visually. Portable and affordable PCR machines like
the miniPCR® thermocycler are being deployed for small-scale SARS-CoV-2 nucleic acid detection
testing. While this method doesn’t have the quantitative precision of an integrated qPCR machine, it
allows diagnosticians to accurately determine if a patient is infected with SARS-CoV-2, pretty much
anywhere.
Diagnostic tests
It’s important to remember that no diagnostic test is perfect, and even highly accurate tests have their limits. For
example, when testing for a virus, two important factors are how much virus was present in the patient sample and the
sensitivity of the diagnostic test.
The amount of SARS-CoV-2 virus present in a patient sample often depends on how long a person has been infected.
For example, if someone has been infected with the SARS-CoV-2 virus very recently, there won’t be very much virus
present because the virus hasn’t had enough time to spread and replicate. For this reason, it’s often recommended to
wait three to five days after a possible exposure to be tested for COVID-19.
The sensitivity of the diagnostic test depends on the type of test being used. Different testing technologies have diferent
detection limits, but qPCR is one of the most sensitive. When you get a COVID-19 test, after they swab your nose, the
swab is placed in a vial full of storage buffer. A qPCR test is able to detect as little as 100 copies of viral genetic material
per milliliter of storage buffer. To get a sense of how sensitive a COVID qPCR test is, it helps to know that patient swabs
that yield positive qPCR COVID-19 tests usually have thousands to billions of copies of viral genetic material per milliliter
of storage buffer.
Diagnosticians refer to the lower bound of the sensitivity of the diagnostic test as the limit of detection. If you are
infected with a very small amount of virus that is below the limit of detection, it is possible to get a negative test result
even though you are actually infected. On the other hand, if the virus has progressed enough to where it is widespread
and in high numbers, the test is expected to always return a positive result.
Understanding both the time from infection and the test’s sensitivity helps a diagnostician accurately interpret results.
But some cases, where the sample sits right at the limit of detection, may yield unclear test results. In these cases, test
results will be reported as indeterminate, or inconclusive, and the test will likely need to be repeated. Being clear about
what diagnostic tests can and cannot detect, and accurately reporting indeterminate or inconclusive results, can help
everyone better interpret test results.
Student’s Guide
Today’s lab
The speed and availability of international travel make it much easier for viral infections to spread
globally and become full-blown pandemics. One way to contain viral infections is to test travelers
when they enter a country so they can quarantine if they arrive infected. Because symptoms for
many diseases are similar, it is not always easy to untangle whether a patient has a run-of-the-mill
cold, seasonal allergies, or a dangerous virus. Further, with many viruses, including SARS-CoV-2,
a patient may be contagious before they show symptoms, or may even be asymptomatic for the
entirety of their infection.
You work at a pop-up screening facility at a small international airport where all incoming patients
are tested for SARS-CoV-2. Your latest incoming flight includes a family in which one member
developed COVID-like symptoms during their 12-hour international flight. You will use molecular
techniques to determine whether anyone in the family is infected with the SARS-CoV-2 virus.
You will test for SARS-CoV-2 infection by using PCR to amplify a ~400 base-pair portion of a SARS-
CoV-2 gene that encodes one of the proteins that makes up the viral coat. The patient samples you
will be given were prepared as follows. A technician first took a nasal swab to collect a sample from
the patient’s airways. RNA was extracted from the sample, and reverse transcription was used to
convert RNA to DNA (Figure 3). You will perform the last step of the workflow and perform PCR
with a fluorescent dye to determine if SARS-CoV-2 genetic material is present in any of the patient
samples.
In addition, you will test two important control samples. The first is referred to as a positive control.
A positive control is a sample where you expect to get a positive result and allows you to assess the
validity of your test. You will use a synthetic segment of the viral gene that the PCR is designed to
detect. If your PCR does not detect SARS-CoV-2 in the positive control sample, you know something
is wrong with the test itself and that the test is invalid.
The second control you will include is referred to as a negative control. A negative control is a
sample where you expect to get a negative result and allows you to test for contamination. Because
PCR is very sensitive and can detect small amounts of viral genetic material, even trace amounts of
viral genetic material that are accidentally introduced into a patient’s sample will be detected. This
could happen if your lab tools became contaminated from a previous positive test. In this case, even
though the patient is actually uninfected, their test result will suggest the presence of viral genetic
material. Including a negative control sample that is known to lack viral genetic material can help
scientists identify when contamination has occurred and the patient tests need to be repeated.
You will use ultra-pure water that does not contain any DNA or RNA as a negative control. If your
PCR detects SARS-CoV-2 in the negative control sample, you know that your samples may be
contaminated.
Student’s Guide
To determine whether any of your patients are infected with the SARS-CoV-2 virus, you will either:
• Collect endpoint data: If your teacher instructs you to follow this protocol, you will
compare the relative fluorescence levels between your four patient samples and your two
control samples once the PCR is complete.
OR
• Collect qPCR time point data: If your teacher instructs you to follow this protocol, you
will measure the relative fluorescence levels between your four patient samples and your
two control samples at several designated times during the PCR.
Both methods will allow you to diagnose your patients for infection with the SARS-CoV-2 virus. The
second method has the added benefit that you will be able to compare the relative viral loads
between samples, but it requires more time.
Patient descriptions
-
Your patients are members of the same household and have just returned from a three-week
international trip.
Patient AH is a 45-year-old male. He presents with a sore throat and a runny nose, but no fever. His
symptoms began shortly after takeoff on a 12-hour international flight. Patient AH is generally in
good health.
Patient BH is a 45-year-old female. She has no symptoms. Patient BH is generally in good health.
Patient CH is a 10-year-old female. She has no symptoms. Patient CH is in good health. Patients CH
and DH are twins.
Patient DH is a 10-year-old male. He has no symptoms. Patient DH is in good health. Patients CH and
DH are twins.
Student’s Guide
Laboratory guides
Note: The data for this lab can be collected two ways: endpoint observation or qPCR time point
observations.
Laboratory guide 1: Endpoint observation
Protocol for endpoint observation P.22
Pre-lab study questions P.28
Post-lab study questions P.33
CER table P.35
Laboratory guide 2: qPCR time point observations
Protocol for qPCR time point observations P.37
Pre-lab study questions P.45
CER table P.51
Laboratory guide 3: Optional gel electrophoresis
Protocol for gel electrophoresis P.53
Student’s Guide
Laboratory guide 1: Protocol for

endpoint observation
Protective gloves and eyewear should be worn for the entirety of this experiment.
PCR setup
-
1. Label 6 PCR tubes (200 μl tubes)
• You should receive a strip of 8 tubes, but
you will only need 6 of them.
• Label each tube on the upper portion of
the sidewall of the tube.
1 2 3 4 5 6
• Note: Writing on the cap of the tube or
the lower half of the sidewall is likely to
rub off later.
2. Add PCR reagents to each tube
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6
2X qGRN 15 μl 15 μl 15 μl 15 μl 15 μl 15 μl
Master Mix
COVID Lab 7.5 μl 7.5 μl 7.5 μl 7.5 μl 7.5 μl 7.5 μl

Primers
DNA sample Patient Patient Patient Patient Positive Negative

AH BH CH DH Control Control
7.5 μl 7.5 μl 7.5 μl 7.5 μl 7.5 μl 7.5 μl
TOTAL 30 μl 30 μl 30 μl 30 μl 30 μl 30 μl
VOLUME
Note: 2X qGRN Master MixTM contains

• Taq DNA polymerase • dNTPs
• PCR buffer with Mg2+ • DNA-binding fluorescent dye
Use a micropipette to add each of

Primer
DNA
MM
the reagents. Remember to change

tips at each step!
Student’s Guide
Flick to mix
3. Cap the tubes and ensure the reagents mix well

• Flick each tube to ensure proper mixing.
• Gently tap the tubes on your bench to ensure
that the liquid collects at the bottom.
Tap to collect liquid at bottom
4. Place the tubes inside the miniPCR® machine

• Press firmly on the tube caps to ensure a tight fit.
• Close the PCR machine lid and tighten it gently.
Student’s Guide
P. 10
PCR programming
-
These instructions are illustrated using miniPCR® software on a Windows PC. Software interfaces
vary slightly by operating system. See the miniPCR® User’s Guide for more details.
If using a different thermal cycler, PCR protocol parameters should remain the same (step 7).
1. Open the miniPCR® app and remain on the “Library” window
2. Connect your miniPCR® thermal cycler to your device using the supplied USB cable or via
Bluetooth®
• Note: Bluetooth® is only available on certain models. To connect via Bluetooth®, select
the icon, located by “Devices” at the left of the desktop app or at the top of the
mobile app.
3. Make sure your miniPCR® thermal cycler is plugged in and that the power switch is turned
on
• Note: If your machine begins running a previously loaded protocol, you may stop it by
clicking or tapping the “X” symbol in the top left box of the “Monitor” window.
4. While in the “Library” window, click the button to create a new protocol
• Button is located in the upper right hand corner of the window.
5. Select “PCR” from the drop-down menu
6. Enter a name for the protocol; for example:

“COVID Lab”
7. Enter the PCR protocol parameters:

• Initial denaturation 94°C, 15 sec
• Denaturation 94°C, 3 sec
• Annealing 58°C, 3 sec
• Extension 72°C, 4 sec
• Number of cycles 25
• Final extension 72°C, 15 sec
Note: The “Heated lid” slider should be in

the on position.
Student’s Guide
8. Click “Save and run” to start the protocol

• If connected to more than one machine, choose the serial number of the miniPCR®
thermal cycler you are using. If asked “Do you want to stop the current protocol…?”, click
“Yes”.
• The lights on the front of the miniPCR® thermal cycler will blink 3 times to indicate that
the protocol has been loaded.
• Note: If needed, you may unplug the USB cable or disconnect Bluetooth® once the
protocol has been loaded. Even if disconnected from your device, the protocol will
continue to completion as normal.
9. Choose “Monitor” window

• The “Monitor” window can be selected on the left column in the desktop app and at the
top in mobile app.
• If more than one miniPCR® thermal cycler is connected to the same device, choose which
machine you would like to monitor using the tabs at the top of the window (desktop
app) or bottom of the Library (mobile app).
The miniPCR® software allows each lab group to monitor the reaction parameters in real time.
.
10. To collect data, observe your samples at 72°C
• You can either:
a. Monitor the PCR reactions and pause the program during the final extension and
observe fluorescence.
OR
b. Wait until the PCR run has completed (approximately 35 min) then heat the samples
to 72°C and observe fluorescence.
• Detailed instructions are provided on the pages 26-27.
Student’s Guide
Data collection
-
Important note: All samples, including the negative control, will be fluorescent at room
temperature. Samples must be observed at 72°C.
• Room temperature is cool enough that the primers used to amplify the DNA will bind to
each other to create double-stranded DNA even though they are not complementary.
The DNA dye will bind to these primer duplexes and emit fluorescence.
• You must heat your samples to 72°C before data collection. At this temperature, the
primers in solution will be single stranded, while the newly synthesized DNA will remain
double stranded.
Data collection during final extension
1. Press the Pause button as soon as the machine enters the final extension stage
• The machine will heat the samples to 72°C and then hold at that temperature.
• Allow your samples to remain at 72°C for at least 10 seconds.
• Pressing pause before miniPCR® reaches 72°C is OK. If the machine has entered the
extension phase, but has not yet reached 72°C, it will continue heating until temperature
is reached and will pause at that time.
• If you press pause too late in the cycle, the program will end. In this case, heat the
samples to 72°C using a miniPCR® in heat block mode or any other heat block.
2. Proceed to step 3 on the next page
Student’s Guide
Data collection after completion of PCR
1. Let the PCR program run to completion (approximately 35 minutes)

• The app status will show “Finished” and the red, yellow, and green LEDs on your
miniPCR® thermal cycler will light up and stay on.
• PCR product is stable at room temperature for several days. For longer term storage,
move tubes to a fridge or freezer.
2. Heat samples for 1 minute at 72°C, then proceed to step 3

• Use a miniPCR® in heat block mode or any other heat block.
3. Open the miniPCR® machine and remove your samples
Be careful not to touch the metal lid which may still be hot
4. Quickly place the tubes in the P51™ molecular fluorescence viewer or other blue light
illuminator
• Immediately record your observations in the data table (Page 33).
• Record your data using the following semi-quantitative scale:
1- no fluorescence / 1- dim fluorescence / 2- moderate fluorescence / 3- full fluorescence
• Use the positive and negative controls as a reference:
° The positive control is expected to be 3- full fluorescence
° The negative control is expected to be 0- no fluorescence
• Remember that as the samples cool, the primers can bind non-specifically and all the
samples will fluoresce. If the negative control begins to fluoresce, just heat the samples
again for 10 seconds at 72°C before trying to record your results.
Student’s Guide
Pre-lab study questions

Review
1. What is the difference between SARS-CoV-2 and COVID-19?
2. What are the two main parts of a virus?
3. Which part of a virus does qPCR detect?
4. In diagnosing a patient with a viral infection, why is it better to use a nucleic acid detection
test than to make a diagnosis based only on the patient’s symptoms?
5. Explain why a reverse transcription step needs to be performed before using qPCR to detect
SARS-CoV-2 infection.
Student’s Guide
6. List two reasons why qPCR is considered the gold standard for detecting SARS-CoV-2
infection.
7. Differentiate between a positive control and a negative control.
Student’s Guide
Critical thinking
1. According to the Centers for Disease Control and Prevention (CDC): People with COVID-19
have had a wide range of symptoms reported—ranging from mild symptoms to severe
illness. Symptoms may appear 2-14 days after exposure to the virus. Anyone can have mild
to severe symptoms.
People with these symptoms may have COVID-19:
• Fever or chills
• Cough
• Shortness of breath or difficulty breathing
• Fatigue
• Muscle or body aches
• Headache
• New loss of taste or smell
• Sore throat
• Congestion or runny nose
• Nausea or vomiting
• Diarrhea
Based on this information, do you think any of your patients are infected with SARS-CoV-2?
How confident are you in your diagnoses?
2. You will carry out a nucleic acid detection test on samples from each of your patients. The
samples will contain genetic material from different sources: from the patients’ own cells,
from bacteria present in the nasal passage, and potentially from viruses, too. Explain how
you can be sure that you are testing specifically for SARS-CoV-2.
Student’s Guide
3. qPCR combines PCR and fluorescence detection. Summarize how fluorescence can be used
to determine if a sample contains genetic material from the SARS-CoV-2 virus.
4. Recall that the brightness of the fluorescence in this PCR test is proportional to the amount
of viral genetic material in the patient’s sample. Imagine one patient’s sample is fluorescent
at the end of the PCR test, but not as brightly fluorescent as the positive control sample.
How would you diagnose this patient? Explain your reasoning.
5. In diagnostic testing, there are two possible types of incorrect test results. A false negative
is a result that incorrectly indicates an absence of infection, whereas a false positive is a
result that incorrectly indicates the presence of an infection.
a. In your opinion, is it more dangerous to have a false negative or a false positive result
when testing for SARS-CoV-2 infection? In which case are the potential consequences
worse? Justify your answer.
b. Which experimental control that you will test can guard against a false negative test
result? Explain your reasoning.
Student’s Guide
c. Which experimental control that you will test can guard against a false positive test
d. Another control typically performed when testing for viral infection is to amplify a
human gene that should be present in all patient samples since we inevitably collect
some of the patients’ own cells along with the viral material. Does this control guard
against false negative or false positive results? Explain your reasoning.
Student’s Guide
Post-lab study questions for

endpoint observation
-
Interpreting results
1. Record your data using the following semi-quantitative scale:

Use the positive and negative controls as a reference:
• The positive control is expected to be 3- full fluorescence
• The negative control is expected to be 0- no fluorescence
Patient Patient Patient Patient Positive Negative

2. After reviewing the results of the experiment, how would you diagnose your patients?
Explain your reasoning.
Patient AH:
Patient BH:
Patient CH:
Patient DH:
Student’s Guide
3. How certain are you of your diagnoses? Explain your reasoning.
Critical thinking
4. What measures do you think the members of this family should take in light of their
diagnoses? Explain your reasoning.
Student’s Guide
CER Table
Fill in the table based on your results from the lab. Use the rubric on the next page to guide your
answers.
Question:
Are any of your patients infected with SARS-CoV-2?
Claim
Make a clear statement
that answers the above question.
Evidence
Provide data from the lab
that supports your claim.
Reasoning
Explain clearly why the data you
presented supports your claim.
Include the underlying scientific
principles that link your evidence
to your claim.
Student’s Guide
Score 4 3 2 1
CLAIM Makes a clear, Makes an accurate Makes an accurate Makes a claim that is
A statement accurate, and and complete claim. but incomplete or inaccurate.
that answers the complete claim. vague claim.
original question/
problem.
EVIDENCE All of the evidence Provides evidence Provides relevant Only provides
Data from the presented is highly that is relevant and but insufficient evidence that does
experiment that relevant and clearly sufficient to support evidence to support not support claim.
supports the sufficient to the claim. the claim. May
claim. support the claim. include some non-
Data must be relevant evidence.
relevant and
sufficient to
support the
claim.
REASONING Provides reasoning Provides reasoning Provides reasoning Provides reasoning

Explain why that clearly links that links the that links the that does not link
your evidence the evidence to evidence to the evidence to the the evidence to
supports your the claim. Relevant claim. Relevant claim, but does not the claim. Does not
claim. This must scientific principles scientific principles include relevant include relevant
include scientific are well integrated are discussed. scientific principles scientific principles
principles/ in the reasoning. or uses them or uses them
knowledge that incorrectly. incorrectly.
you have about
the topic to
show why the
data counts as
evidence.
We recommend that teachers use the following scale when assessing this assignment using the rubric.
Teachers should feel free to adjust this scale to their expectations.
Rubric score 3 4 5 6 7 8 9 10 11 12
Equivalent Grade 55 60 65 70 75 80 85 90 95 100
Student’s Guide
Laboratory guide 2: Protocol for

qPCR time point observations
Protective gloves and eyewear should be worn for the entirety of this experiment.
PCR setup
-
1. Label 6 PCR tubes (200 μl tubes)
• You should receive a strip of 8 tubes, but
you will only need 6 of them.
• Label each tube on the upper portion of
the sidewall of the tube. 1 2 3 4 5 6
• Note: Writing on the cap of the tube or
the lower half of the sidewall is likely to
rub off later.
2. Add PCR reagents to each tube
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6
2X qGRN 15 μl 15 μl 15 μl 15 μl 15 μl 15 μl
Master Mix
COVID Lab 7.5 μl 7.5 μl 7.5 μl 7.5 μl 7.5 μl 7.5 μl

Primers
DNA sample Patient Patient Patient Patient Positive Negative

7.5 μl 7.5 μl 7.5 μl 7.5 μl 7.5 μl 7.5 μl
TOTAL 30 μl 30 μl 30 μl 30 μl 30 μl 30 μl
VOLUME
Note: 2X qGRN Master MixTM contains:

• Taq DNA polymerase • dNTPs
• PCR buffer with Mg2+ • DNA-binding fluorescent dye
Use a micropipette to add each of

Primer
DNA
MM
the reagents. Remember to change

tips at each step!
Student’s Guide
Flick to mix
3. Cap the tubes and ensure the reagents mix well

• Flick each tube to ensure proper mixing.
• Gently tap the tubes on your bench to ensure
that the liquid collects at the bottom.
Tap to collect liquid at bottom
4. Place the tubes inside the miniPCR® machine

• Press firmly on the tube caps to ensure a tight fit.
• Close the PCR machine lid and tighten it gently.
Student’s Guide
P. 10
PCR programming
-
These instructions are illustrated using miniPCR® software on a Windows PC. Software interfaces
vary slightly by operating system. See the miniPCR® User’s Guide for more details.
If using a different thermal cycler, PCR protocol parameters should remain the same (step 7).
1. Open the miniPCR® app and remain on the “Library” window
2. Connect your miniPCR® thermal cycler to your device using the supplied USB cable or via
Bluetooth®
• Note: Bluetooth® is only available on certain models. To connect via Bluetooth®, select
the icon, located by “Devices” at the left of the desktop app or at the top of the
mobile app.
3. Make sure your miniPCR® thermal cycler is plugged in and that the power switch is turned
on
• Note: If your machine begins running a previously loaded protocol, you may stop it by
clicking or tapping the “X” symbol in the top left box of the “Monitor” window.
4. While in the “Library” window, click the button to create a new protocol
• Button is located in the upper right hand corner of the window.
5. Select “PCR” from the drop-down menu
6. Enter a name for the protocol; for example:

“COVID Lab”
7. Enter the PCR protocol parameters:

• Initial denaturation 94°C, 15 sec
• Denaturation 94°C, 3 sec
• Annealing 57°C, 3 sec
• Extension 72°C, 4 sec
• Number of cycles 25
• Final extension 72°C, 15 sec
Note: The “Heated lid” slider should be in

the on position.
Student’s Guide
Instructions for collecting data throughout the PCR

Important note: All samples, including the negative control, will be fluorescent at room
temperature. Samples must be observed at 72°C.
• Room temperature is cool enough that the primers used to amplify the DNA will bind to
each other to create double-stranded DNA even though they are not complementary.
The DNA dye will bind to these primer duplexes and emit fluorescence.
• You must heat your samples to 72°C before data collection. At this temperature, the
primers in solution will be single stranded, while the newly synthesized DNA will remain
double stranded.
• You will pause the PCR program during the “extension” step of a PCR cycle when
samples are at 72°C.
• Each step in the PCR cycle (denaturation, annealing, extension) is less than 5 seconds
long, so you need to carefully monitor the reaction to make sure you pause the machine
and collect data at the correct time.
• You will collect data during cycle 1, then every three cycles starting with cycle 10.
1. Click “Save and run” to start the protocol

• If asked “Do you want to stop the current protocol…?”, click “Yes”.
• The lights on the front of the miniPCR® thermal cycler will blink 3 times to indicate that
the protocol has been loaded.
2. Choose “Monitor” window

• The “Monitor” window can be selected on the left column in the desktop app and at the
top in mobile app.
Continued on the next page
Student’s Guide
Initial observation at cycle 1
3. Press the Pause button as soon as the machine enters the extension stage
• Allow your samples to remain at 72°C for at least 5 seconds before removing.
• Open the miniPCR® machine and remove your samples.
• Pressing pause before miniPCR® reaches 72°C is OK. If the machine has entered the extension
phase, but has not yet reached 72°C, it will continue heating until temperature is reached and
will pause at that time.
• If you press pause too late in the cycle, the machine will continue on to the denaturation stage
and hold at 94°C. If this happens, your samples will denature and no fluorescence will be
visible. Skip this cycle and measure at the next extension step.
Student’s Guide
4. Quickly place the tubes in the P51™ molecular

fluorescence viewer or other blue light illuminator
• Immediately record your observations on the
‘cycle 1’ row of the data table (Page 44).
• Record your data using the following semi-
quantitative scale:
0- no fluorescence
1- dim fluorescence
2- moderate fluorescence
3- full fluorescence
• Remember that as the samples cool, the
primers can bind non-specifically and all the
samples will fluoresce. If this happens, just put
the samples back in the PCR machine at 72°C
for 10 seconds before trying to record your
results.
5. Return the tubes to the miniPCR® machine and press the Run button
Subsequent observations starting at cycle 10
6. Allow the PCR to continue until cycle 10

• It will take approximately 10 minutes to reach cycle 10 of your PCR protocol.
• Keep an eye on the progress of your PCR. You don’t want to miss your observation time
point!
Student’s Guide
7. Once the PCR reaches cycle 10, press the Pause button as soon as the machine enters the
extension stage
• Allow your samples to remain at 72°C for at least 5 seconds before removing.
• Open the miniPCR® machine and remove your samples.
8. Quickly place the tubes in the P51™ molecular fluorescence viewer or other blue light
illuminator
• Immediately record your observations on the ‘cycle 10’ row of the data table on the next
page.
9. Return the tubes to the miniPCR® machine and press the Run button
Student’s Guide
10. Repeat steps 7-9 to collect data according to the chart below:
Fluorescence level at 72˚C
Cycle # Patient Patient Patient Patient Positive Negative

10
13
16
19
22
25
• Record your data using the following semi-quantitative scale:

• Remember that as the samples cool, the primers can bind non-specifically and all the
samples will fluoresce. If this happens, just put the samples back in the PCR machine at
72°C for 10 seconds before trying to record your results.
11. When the PCR run has completed, app status will show “Finished” and the red, yellow, and
green LEDs on your miniPCR® thermal cycler will light up and stay on
12. PCR product is stable at room temperature for several days. For longer term storage, move
tubes to a fridge or freezer
• Tubes may remain inside the miniPCR® thermal cycler for several days following protocol
completion.
Student’s Guide
Pre-lab study questions

Review
1. What is the difference between SARS-CoV-2 and COVID-19?
2. What are the two main parts of a virus?
3. Which part of a virus does qPCR detect?
4. In diagnosing a patient with a viral infection, why is it better to use a nucleic acid detection
test than to make a diagnosis based only on the patient’s symptoms?
5. Explain why a reverse transcription step needs to be performed before using qPCR to detect
SARS-CoV-2 infection.
Student’s Guide
6. List two reasons why qPCR is considered the gold standard for detecting SARS-CoV-2
infection.
7. Differentiate between a positive control and a negative control.
Student’s Guide
Critical thinking
1. According to the Centers for Disease Control and Prevention (CDC): People with COVID-19
have had a wide range of symptoms reported—ranging from mild symptoms to severe
illness. Symptoms may appear 2-14 days after exposure to the virus. Anyone can have mild
to severe symptoms.
People with these symptoms may have COVID-19:
• Fever or chills
• Cough
• Shortness of breath or difficulty breathing
• Fatigue
• Muscle or body aches
• Headache
• New loss of taste or smell
• Sore throat
• Congestion or runny nose
• Nausea or vomiting
• Diarrhea
Based on this information, do you think any of your patients are infected with SARS-CoV-2?
How confident are you in your diagnoses?
2. You will carry out a nucleic acid detection test on samples from each of your patients. The
samples will contain genetic material from different sources: from the patients’ own cells,
from bacteria present in the nasal passage, and potentially from viruses, too. Explain how
you can be sure that you are testing specifically for SARS-CoV-2.
Student’s Guide
3. qPCR combines PCR and fluorescence detection. Summarize how fluorescence can be used
to determine if a sample contains genetic material from the SARS-CoV-2 virus.
4. In diagnostic testing, there are two possible types of incorrect test results. A false negative
is a result that incorrectly indicates an absence of infection, whereas a false positive is a
result that incorrectly indicates the presence of an infection.
a. In your opinion, is it more dangerous to have a false negative or a false positive result
when testing for SARS-CoV-2 infection? In which case are the potential consequences
worse? Justify your answer.
b. Which experimental control that you will test can guard against a false negative test
c. Which experimental control that you will test can guard against a false positive test
Questions continue on next page.
Student’s Guide
d. Another control typically performed when testing for viral infection is to amplify a
human gene that should be present in all patient samples since we inevitably collect
some of the patients’ own cells along with the viral material. Does this control guard
against false negative or false positive results? Explain your reasoning.
Student’s Guide

-
Interpreting results 3
1. Graph your data. Be sure to make 2.5
your six data sets look different

by using a different color or style 2
Fluorescence
Legend
of data point each sample (AH, AH
BH, etc.). Indicate which symbols 1.5

BH
CH
DH
represent which samples in the + control
- control
graph legend. 1
0.5
2. After reviewing the results of
the experiment, how would you
0
diagnose your patients? Explain 1 4 7 10 13 16 19 22 25
PCR cycle number

your reasoning.
Patient AH:
Patient BH:
Patient CH:
Patient DH:
3. Can you draw any conclusions about the relative viral loads in your patients? Explain your
reasoning.
4. What measures do you think the members of this family should take in light of their
diagnoses? Explain your reasoning.
Student’s Guide
CER Table
Fill in the table based on your results from the lab. Use the rubric on the next page to guide your
answers.
Question:
Are any of your patients infected with SARS-CoV-2?
Claim
Make a clear statement
that answers the above question.
Evidence
Provide data from the lab
that supports your claim.
Reasoning
Explain clearly why the data you
presented supports your claim.
Include the underlying scientific
principles that link your evidence
to your claim.
Student’s Guide
Score 4 3 2 1
CLAIM Makes a clear, Makes an accurate Makes an accurate Makes a claim that is
A statement accurate, and and complete claim. but incomplete or inaccurate.
that answers the complete claim. vague claim.
original question/
problem.
EVIDENCE All of the evidence Provides evidence Provides relevant Only provides
Data from the presented is highly that is relevant and but insufficient evidence that does
experiment that relevant and clearly sufficient to support evidence to support not support claim.
supports the sufficient to the claim. the claim. May
claim. support the claim. include some non-
Data must be relevant evidence.
relevant and
sufficient to
support the
claim.
REASONING Provides reasoning Provides reasoning Provides reasoning Provides reasoning

Explain why that clearly links that links the that links the that does not link
your evidence the evidence to evidence to the evidence to the the evidence to
supports your the claim. Relevant claim. Relevant claim, but does not the claim. Does not
claim. This must scientific principles scientific principles include relevant include relevant
include scientific are well integrated are discussed. scientific principles scientific principles
principles/ in the reasoning. or uses them or uses them
knowledge that incorrectly. incorrectly.
you have about
the topic to
show why the
data counts as
evidence.
We recommend that teachers use the following scale when assessing this assignment using the rubric.
Teachers should feel free to adjust this scale to their expectations.
Rubric score 3 4 5 6 7 8 9 10 11 12
Equivalent Grade 55 60 65 70 75 80 85 90 95 100
Student’s Guide
Laboratory Guide 3: Protocol for

Gels can be prepared up to three days ahead of time and stored at

ambient temperature, covered in air-tight plastic wrap and protected
from light.
You will need 6 lanes plus one lane for ladder per group. It is possible
for groups to share a gel by using two combs.
These instructions are designed for use with the blueGel™ electrophoresis system by miniPCR
bio™. If using another electrophoresis system, these instructions may need to be adjusted according
to the manufacturer’s instructions.
1. Prepare 1X TBE buffer (to be completed by teacher in advance)

• TBE buffer is often provided as liquid concentrate or powder.
• Follow manufacturer’s instructions to prepare 1X TBE buffer solution.
Comb
2. Prepare a clean and dry casting platform with a gel
tray and comb Gel tray
• Place the clear gel tray in the white casting
platform.
• Place a well-forming comb at the top of the
gel tray.
3. Prepare a 2% agarose solution with a fluorescent Casting

DNA stain (e.g., SeeGreenTM or GelGreen®) using platform
the method indicated by your instructor
IMPORTANT NOTE: There are several ways to prepare agarose gels.
• Scan the QR code for detailed instructions on how to prepare

agarose gels.
• Both written and video instructions are available.
www.minipcr.com/agarose-gel/
Student’s Guide
4. Pour the agarose solution into the prepared casting platform with a gel tray and comb
• The agarose solution should cover the bottom of the gel tray and the bottom 3 mm of
the comb (roughly the bottom 1/3 of the comb).
5. Allow gel to solidify completely and remove the comb

by pulling firmly upwards
• Gels will typically be ready in about 10 minutes.
• Gel is ready when cool and firm to the touch.
Student’s Guide
Gel electrophoresis - Running the gel

These instructions are designed for use with blueGel™ electrophoresis system by miniPCR bio™. If
using another electrophoresis system, these instructions may need to be adjusted according to the
manufacturer’s instructions.
1. Place the gel tray containing your gel in the buffer

Gel tray
chamber
• Ensure that the clear buffer chamber is inside the
Buffer
blueGel™ electrophoresis system. chamber
• The wells of the gel should be on the same side

as the negative electrode, away from the power Base
button.
2. Add 30 ml of 1X TBE electrophoresis buffer

• The buffer should just cover the gel and fill the wells.
• Ensure that there are no air bubbles in the wells
(shake the gel gently if bubbles need to be
dislodged).
3. Add loading dye to your samples

• Add 4 μl of loading dye to each tube.
• Pipette up and down until thoroughly mixed.
Note: Change pipette tips between samples to prevent
contamination.
4. Load samples onto the gel in the following sequence

• Lane 1: 10 μl Fast DNA Ladder 1
• Lane 2: 15 μl Patient AH PCR product
• Lane 3: 15 μl Patient BH PCR product
• Lane 4: 15 μl Patient CH PCR product
• Lane 5: 15 μl Patient DH PCR product
• Lane 6: 15 μl Positive Control PCR product
• Lane 7: 15 μl Negative Control PCR product
Note: Change pipette tips between samples to prevent
contamination.
Student’s Guide
5. Place the orange cover on the blueGel™ electrophoresis

system
• To prevent fogging, make sure that ClearView™ spray has
been evenly applied to the inside of the orange cover.
• Match the positive and negative electrode signs on the
orange lid with the corresponding positive and negative
signs on the blue base.
• The electrodes of the lid should be aligned with the metal
leads on the base.
• The orange lid should sit flush with the blue base using
little force.
6. Press the “Run” button

• Check that the green light beside the power button remains illuminated.
7. Conduct electrophoresis for 15-25 minutes

• Note: Check the progress of your samples every 10 minutes to monitor the migration of
your DNA samples.
• Longer electrophoresis times will result in better separation of similar sized DNA
fragments. However, if run too long, small DNA fragments can run off the end of the gel
or lose fluorescence.
Student’s Guide
Gel electrophoresis – Visualizing

results
1. Press the “light bulb” button to turn on the blueGel™
transilluminator
• For best viewing, dim lights or use Fold-a-View™ photo
documentation hood with a smartphone camera.
• Gels may be viewed at the end of the run or
periodically throughout the run.
• If the image appears hazy, wipe off the inside of the Fast DNA bp
Ladder 1
orange cover and reapply ClearView™ spray.
1200
2. Ensure that there is sufficient DNA band resolution
• Run the gel longer if needed to increase resolution. 700
500
3. Document your results
300
• Place Fold-a-View™ photo documentation hood on the
blueGel™ electrophoresis system to take a picture with 200
a smartphone or other digital camera. 100

• Compare the bands from the DNA samples to the
ladder to obtain size estimates.
Student’s Guide

-
Interpreting results
1. Use the image on the right to illustrate your gel

electrophoresis results. There are seven lanes
on the gel: one for your ladder, and one for
each sample. Be sure to label each lane with the
source of the sample.
2. Do the results of your gel confirm the diagnoses

you made based on fluorescence? Explain your
reasoning.
3. What information can you get from your gel that you could not get from observing your
reactions in a tube?
Extension
Activities
Quantification with qPCR P.60
Additional background: COVID testing P.62
Additional
Supports
Extension: Quantification with qPCR

qPCR machines contain a built-in sensor that measures the fluorescence of every sample after each
PCR cycle. When you assess the fluorescence after each PCR cycle, you can determine how many
cycles it takes for the fluorescence to be detectable. The cycle where fluorescence is first observable
above background levels is called Ct, for cycle threshold.
How soon a reaction reaches the threshold is directly related to how much of the target sequence
was in the sample at the starting point. The more target DNA there is to begin with, the sooner Ct
will be reached. This means that by observing Ct, we can learn about our original DNA concentra-
tion. Because the amount of target DNA doubles every cycle, if reaction A reaches Ct one cycle
earlier than reaction B, you can estimate that reaction A started with roughly twice as much target
sequence as reaction B. If reaction A reaches Ct two cycles before reaction C, you can estimate that
there was a four-fold difference in the original amount of the target sequence, and so on.
The following graph represents the relative fluorescence of three different samples during a qPCR
experiment. Use the information in the graph to answer the questions below.
Relative fluorescence
Sample 1
Sample 2
Sample 3
PCR cycle number
1. Record the Ct for each sample in the table below:
Sample Ct
Additional
Supports
2. Which sample contained the greatest concentration of the target DNA? Explain your
reasoning, and be sure to reference the Ct for the sample in your answer.
3. Which sample contained the lowest concentration of the target DNA? Explain your
reasoning, and be sure to reference the Ct for the sample in your answer.
4. What is the relative difference in the starting concentration of the target DNA between
Sample 2 and Sample 3? You may use a calculator, but show your work.
5. For some viruses, determining the number of viral particles present in a patient, or viral
load, is important in determining the best course of treatment. The typical COVID-19 test is
done in a qPCR machine and therefore will report a Ct value for each patient, even though
the result is usually just reported as positive or negative. The Ct value corresponds to viral
load, and researchers are currently comparing Ct values to patient outcomes for COVID-19
patients.
What would be your hypothesis about the relationship between a COVID-19 patient’s Ct
value and the severity of their disease? Explain your reasoning.
Additional
Supports
Additional background: COVID-19 testing

There are numerous lab tests that doctors use to diagnose COVID-19, and the testing landscape can
change rapidly as new tests are developed or testing guidelines are updated. Refer to the Food and
Drug Administration for the most up-to-date information on COVID-19 testing:
https://www.fda.gov/consumers/consumer-updates/coronavirus-disease-2019-testing-basics.
Diagnostic test accuracy

There are two important measures of diagnostic tests: sensitivity and specificity.
Sensitivity is a measure of how well a test can correctly generate a positive test result for people
who do have the condition being tested for. Having a test with high sensitivity is important to
prevent false negative test results. A false negative is a result that incorrectly indicates an absence of
the condition being tested for. For example, if someone received a negative COVID test result, when
in fact, they were infected with the SARS-CoV-2 virus.
Specificity is a measure of how well a test can correctly generate a negative test result for people
who do not have the condition being tested for. Having a test with high specificity is important to
prevent false positive test results. A false positive is a result that incorrectly indicates the presence of
the condition being tested for. For example, if someone received a positive COVID test result, when
in fact, they were not infected with the SARS-CoV-2 virus.
Ideally, a diagnostic test would have both high sensitivity and high specificity, but as sensitivity
increases, specificity typically decreases and vice versa. Selecting the balance between sensitivity
and specificity depends on what the test is being used to diagnose.
Test result
“Positive” “Negative”
Infected
True positive False negative

Actual status
Not infected
False positive True negative
Additional
Supports
Types of COVID-19 tests

There are two general methods for testing whether a person is or has been infected with a virus:
antibody tests and viral tests. Antibody tests detect antibodies in your blood produced in response
to infection with the SARS-CoV-2 virus or a vaccine. Antibody tests will tell you if you have been
exposed to a virus or vaccine in the past, but they cannot be used to diagnose an active infection.
On the other hand, viral tests (also called diagnostic tests) detect an active infection with the SARS-
CoV-2 virus and are used to diagnose patients. There are two main classes of viral tests: nucleic acid
tests and antigen tests. These are described in the table below.
Viral tests (also called diagnostic tests) identify active viral infections. There
are two main types of viral test: nucleic acid tests and antigen tests.
Nucleic acid tests detect viral genetic Antigen tests detect viral antigens,
material molecules on the surface of the virus
How long • As quickly as under an hour if testing • Results typically reported in minutes
does it performed on-site
take to get • More typically a few days, as samples are
results? usually sent to a testing lab
Pros • Additional testing typically not needed to • Faster

verify test results • Easier to perform
• Highly sensitive—can detect early infections • Less expensive
• High specificity • High specificity
Cons • More expensive • Less sensitive—may not detect an early or

• Require expensive specialized equipment late infection
• Additional nucleic acid test may be
required to verify negative test results
Rapid tests (also called point-of-care tests) are diagnostic tests that are performed where the
sample is collected from the patient, with results ready in minutes or hours rather than days. Rapid
tests exist for both nucleic acid tests and antigen tests, although only antigen tests are available for
at home self-testing.
You have probably also heard about variants of the virus that causes COVID-19. Variants are versions
of the virus that have acquired mutations in the viral genome. Some variants may be more infectious
or more deadly than the original form of the virus, so people are often not just concerned with
whether a patient is infected, but also want to know which specific variant of the virus is responsible
for the infection. Standard testing only detects the presence of viral genetic material and cannot
identify which variant of the SARS-CoV-2 virus a person is infected with. To identify variants, viral
genetic material is amplified using PCR, and then typically sequenced. Sequencing reads each of
the nucleic acid bases or building blocks of the viral genetic material, allowing scientists to compare
with other viral sequences and identify variants. Sequencing is usually only performed in certain
cases or only on a select subset of tests so that public health officials may monitor which variants
are spreading in the community.
Instructor’s
Guide
Expected results P.65
Notes on lab design P.67
Differentiation P.67
Additional student supports P.68
Extension activities P.68
Placement in unit P.69
Learning goals and skills developed P.69
Standards alignment P.70
Ordering information P.71
About miniPCR bio Learning Labs™ P.72
Expected results
Endpoint observation
Results should resemble the photo below:
AH BH CH DH + -
Data collected at 72°C after cycle 25
Interpretation
By comparing each patient sample with the controls, students should be able to diagnose the family
as follows:
• Patient AH: Patient AH’s sample is brightly fluorescent, indicating that they are infected with
SARS-CoV-2
• Patient BH: Patient BH’s sample is brightly fluorescent, indicating that they are infected with
SARS-CoV-2
• Patient CH: Patient CH’s sample is brightly fluorescent, indicating that they are infected with
SARS-CoV-2
• Patient DH: Patient DH’s sample is not fluorescent, indicating that they are not infected with
SARS-CoV-2
Note: If students run out of class time before their PCR is done, they can collect initial observations.
• Have students pause the PCR program during the extension step (72°C), then remove samples
and observe using the P51™ molecular fluorescence viewer or other blue light illuminator.
• For detailed instructions on how to pause the PCR program, see page 26.
• Return samples to the thermal cycler and resume the PCR program. Samples can be left
unattended once the PCR program is complete, and are stable for one week at room
temperature.
• Have students collect their final observations the next class period. Briefly heat samples at
72°C then observe using the P51™ molecular fluorescence viewer or other blue light illuminator.
As long as the PCR has completed 20 cycles, samples AH, CH, and the Positive Control are expected
to be brightly fluorescent. Sample BH, which contained a lower concentration of simulated viral DNA
will likely be fluorescent, but at lower levels. After 25 cycles, samples AH, BH, CH are all expected to
display a similar level of fluorescence to the Positive Control.

If your students follow the instructions in protocol 2 to monitor their reactions during
the PCR, results should be similar to what is listed below. Note that because students are rating the
fluorescence by eye, individual results may vary.
• Positive Control: Fluorescence should become detectable around cycle 14-16. Fluorescence will
likely plateau around cycle 22.
• Negative Control: Fluorescence should not be observed for this sample.
• Patients AH and CH: Results should be similar to the Positive Control sample. Fluorescence
should become detectable around cycle 14-16. Fluorescence will likely plateau around cycle 22.
• Patient BH: Fluorescence should become detectable around cycle 18-20. Fluorescence will
likely plateau around cycle 25.
• Patient DH: Fluorescence should not be observed for this sample.
Interpretation
By comparing the endpoint fluorescence of each patient sample with the controls, students can
conclude that patients AH, BH, and CH are infected, while patient DH is uninfected. By comparing
the Ct values, students can conclude that Patient BH has a lower viral load than both Patient AH and
Patient CH.
Optional gel electrophoresis

If your students perform the optional gel electrophoresis, results should resemble the photo below:
l l
d er n tro ntro
d co o
La AH BH CH DH + -c
Base
Pairs
1200
700
500
300
200
100
The expected size of the SARS-CoV-2 PCR product is ~400 bp. This image represents results
obtained after a 20 minute run using a blueGel™.
Interpretation
By comparing each patient sample with the controls, students should be able to confirm that
patients AH, BH, and CH are infected, while patient DH is uninfected.
Notes on lab design

This lab was written during the COVID-19 pandemic in response to high demand for tools to teach
about viral outbreaks in a safe, hands-on way.
Disclaimer: no pathogenic materials are used in this experiment. This experimental protocol
engages students in a simulated patient diagnosis exercise. None of the materials provided in the lab
kit present a pathogenic risk. For educational use only. Not for diagnostic use.
The design of this lab has simplified certain elements to achieve its goals. Some of these elements
include:
• The DNA samples provided consist of plasmid DNA, not viral DNA or animal tissue. References
to viral strains or patient samples are used only to recreate a plausible clinical scenario.
• qPCR can be performed using either fluorescent dyes or using a sequence-specific fluorescent
probe. This lab visualizes PCR products using a dye that fluoresces when bound to any double
stranded DNA, while diagnostic qPCR tests often use a sequence-specific fluorescent probe.
Differentiation
This lab serves as an introductory look at how DNA and biotechnology can be used to solve clinical
problems. With simple modifications, this activity can be used effectively in classes ranging from
general high school through college.
Introductory classes: Focus on scientific practices including critically evaluating different types of
evidence, making predictions, and designing well-controlled experiments. Limit coverage of
genetics to the concept of DNA as a unique identifier for specific pathogens. We recommend that
introductory students perform endpoint observation of their samples once the PCR is complete
(instructions on page 22-27). This approach allows students to diagnose their patients as being
inflected or infected with the SARS-CoV-2 virus without delving into the complexity of qPCR.
Advanced classes: Dive into the principles and practice of qPCR. Use the extension activity,
Quantification with qPCR (page 60) to introduce students to the quantitative power of qPCR, and
have your students collect data throughout the PCR to generate qPCR curves and compare viral
loads (instructions on pages 37-44).
Additional student supports

At miniPCR bio™, we are committed to preparing students to be successful in the laboratory through
high quality curriculum and training. We have created an extensive set of resources to help your
students succeed in molecular biology techniques, all of which are available for free download at the
miniPCR bio™ tutorials page of our website.
-
https://www.minipcr.com/tutorials/.
Those activities most relevant to this lab are listed below.
Micropipetting: Video and activity resources to train students in the basic use of a micropipette.
PCR: Video and worksheet activity instructing students on the fundamentals and practice of PCR.
Gel electrophoresis: Video and worksheet activity instructing students on the fundamentals and
practice of agarose gel electrophoresis.
Visit https://www.minipcr.com/covid-resources/ to access the complete miniPCR bio™ COVID-19

resource library, including:
• mRNA vaccine resources - DNAdots article and webinar
• qPCR resources - DNAdots article and webinar
Extension activities
Quantification with qPCR (page 60): Introduce students to the quantitative power of qPCR.
Additional background: COVID testing (page 62): While qPCR is the gold standard for COVID-19
diagnosis, there are numerous COVID-19 tests. This resource compares the different types of
COVID-19 tests.
Placement in unit
Human health: For classes focused on human health and pathology, this lab introduces students
to infectious disease and diagnostics using an interactive case study format. This lab would offer a
compelling introduction to a unit on the immune or respiratory systems.
DNA and biotechnology: This lab can serve as an engaging and practical context to familiarize
students with diagnostic applications of PCR.
Learning goals and skills developed

Student Learning Goals:
• Use molecular techniques to diagnose fictional patients with a simulated viral infection
• Understand that PCR is a technique for amplifying specific DNA sequences
• Explain how different biological entities can be identified by their unique genetic sequences
• Describe how qPCR can be used to diagnose viral infections
Scientific Inquiry Skills:

• Identify or pose a testable question
• Identify dependent and independent variables and appropriate experimental controls
• Follow detailed experimental protocols
• Create tables or graphs to present their results
• Interpret data presented in a chart or table
• Make a claim based on scientific evidence
• Use reasoning to justify a scientific claim
Molecular Biology Skills:

• Micropipetting
• Principles and practice of PCR
• Use of fluorescence to visualize PCR results
Optional:
• Preparation of agarose gels
• Agarose gel DNA electrophoresis
• Staining, visualization, and molecular weight analysis of DNA fragments
Standards alignment
Next Generation Science Standards
Students who demonstrate understanding can:

HS-LS1-1. Construct an explanation based on evidence for how the structure of DNA determines the structure
of proteins which carry out the essential functions of life through systems of specialized cells.
Science and Engineering Disciplinary Core Ideas Crosscutting Concepts

Practice
• Asking Questions and Defining LS1.A: From Molecules to Organisms: • Patterns
Problems Structures and Processes • Cause and Effect
• Planning and Carrying Out • Scale, Proportion And Quantity
Investigations Structure and Function
• Analyzing and Interpreting Data • Interdependence of Science,
Using Mathematics and Computational Engineering, and Technology
Thinking
• Constructing Explanations and
Designing Solutions
• Engaging in Argument from Evidence
• Obtaining, Evaluating, and
Communicating Information
Common Core ELA/Literacy Standards

RST.9-10.1 Cite specific textual evidence to support analysis of science and technical texts, attending to the
precise details of explanations or descriptions.
RST.9-10.3 Follow precisely a complex multistep procedure when carrying out experiments, taking measurements,
or performing technical tasks, attending to special cases or exceptions defined in the text.
RST.9-10.4 Determine the meaning of symbols, key terms, and other domain-specific words and phrases as they are
used in a specific scientific or technical context relevant to grades 9-10 texts and topics.
RST.9-10.5 Analyze the structure of the relationships among concepts in a text, including relationships among key
terms (e.g., force, friction, reaction force, energy).
RST.9-10.9 Compare and contrast findings presented in a text to those from other sources (including their own
experiments), noting when the findings support or contradict previous explanations or accounts.
WHST.9-10.1 Write arguments focused on discipline-specific content.
WHST.9-10.2 Write informative/explanatory texts, including the narration of historical events, scientific procedures/
experiments, or technical processes.
WHST.9-10.9 Draw evidence from informational texts to support analysis, reflection, and research.
* For simplicity, this activity has been aligned to high school NGSS and grades 9-10 Common Core standards.
Ordering information
To order COVID-19 qPCR Learning Lab kits, you can:
Call (781)-990-8PCR
email us at orders@minipcr.com
visit https://www.minipcr.com
COVID-19 qPCR Learning Lab (catalog no. KT-1900-06) contains the following reagents supplies:
• 2X qGRN Master Mix
• COVID Lab Primers
• Simulated Patient Samples:
° Patient AH
° Patient BH
° Patient CH
° Patient DH
• Simulated Control Samples:
° Positive Control
° Negative Control
• PCR strip tubes
• Fast DNA Ladder 1
• 6X Loading Dye
Disclaimer: no pathogenic materials are used. This experimental protocol engages students in a
simulated patient diagnosis exercise. None of the materials provided in this lab kit pose a pathogenic
risk. For educational use only. Not for diagnostic use.
Materials are sufficient for 8 lab groups, or 32 students

All components should be kept frozen at -20°C for long-term storage
Reagents must be used within 12 months of shipment
Other reagents needed if performing optional gel electrophoresis

Additional reagents can be sourced independently or are available as part of our Learning Lab
Companion Kit (catalog no. KT-1510-01) at miniPCR.com.
• Agarose (electrophoresis grade)
• Fluorescent DNA stain (e.g., SeeGreenTM or GelGreen®)
• Gel electrophoresis buffer (e.g., 1X TBE)
• Distilled or deionized H2O (to dilute TBE buffer concentrate – not included in Lab
Companion Kit)
About miniPCR bio Learning LabsTM

This Learning Lab was developed by the miniPCR bioTM team in an effort to help more students
understand concepts in molecular biology and to gain hands-on experience in real biology and
biotechnology experimentation.
We at miniPCR bioTM believe, based on our direct involvement working in educational settings, that
it is possible for these experiences to have a real impact in students’ lives. Our goal is to increase
everyone’s love of DNA science, scientific inquiry, and STEM. We develop miniPCR Learning LabsTM
to help achieve these goals, working closely with educators, students, academic researchers, and
others committed to science education.
The guiding premise for this lab is that a PCR experiment can recapitulate a real-life biotechnology
application and provide the right balance between intellectual engagement, inquiry, and discussion.
Starting on a modest scale working with Massachusetts public schools, miniPCR bio Learning
LabsTM have been well received, and their use is growing rapidly through academic and outreach
collaborations across the world.

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COVID-19 qPCR Lab

For educational use only. Not for diagnostic use.

COVID-19 qPCR Lab Version: 1.1

TECHNIQUES TOPICS LEVEL WHAT YOU NEED AP CONNECTION

Micropipetting Infectious disease General high Micropipettes AP Biology units 6.8

Planning your time

Option 1: Endpoint observation Option 2: qPCR time point observations

SINGLE CLASS PERIOD: 50 MINUTES SINGLE CLASS PERIOD: 80 MINUTES

Option 1: Endpoint observation

SINGLE CLASS PERIOD: 50 MINUTES

Once PCR has started, samples may be left unattended.

Prepare View and Optional: Gel

* The PCR program will * Students will perform a * Allow an additional 25

Option 2: qPCR time point observations

This option requires:

Run PCR and collect several

COVID Lab Primers 550 μl 55 μl Freezer

Simulated Patient Samples 100 μl each 10 μl each Freezer

Simulated Control Samples 100 μl each 10 μl each Freezer

PCR strip tubes Ten 8-tube One 8-tube Room

For optional gel electrophoresis:

6X Loading Dye 300 μl 25 μl Freezer

Fast DNA Ladder 1 150 μl 15 μl Freezer

Materials needed (cont.)

P51™ molecular fluorescence viewer 1

or other blue light illuminator: Can be shared between groups

Disposable micropipette tips At least 18 per group

PCR tubes (0.2 ml): Included in the Lab 6

Plastic tubes (1.7 ml): Included in the Lab 8

Additional materials needed for

Distilled water for making agarose gels and 50 ml per gel

Heat-proof flask or beaker to dissolve 1 per class

Microwave or hot plate to dissolve agarose 1 per class

Sold separately in Learning Lab Companion Kit

TBE electrophoresis Supplied as liquid 30 ml 1X solution Room temp.

PCR tubes (0.2 ml) 100

Plastic tubes (1.7 ml) 50

Lab setup: PCR and fluorescence

Preparation for PCR Defrost tubes

• Thaw tubes containing 2X qGRN Master

- 2X qGRN Master Mix 110 μl

• Distribute supplies and reagents to lab

groups (continued on next page).

2X qGRN Master Mix 110 μl

COVID Lab Primers 55 µl

PCR tubes (200 μl) 1 strip of 8 tubes

Micropipette tips At least 18

Fine tipped permanent marker 1

6 wells in a miniPCR® or other thermocycler

Access to a P51™ or other blue light illuminator

• Thermal cyclers can be programmed ahead of time

Lab setup: Optional gel

Preparation for gel electrophoresis

PCR products from previous section of the lab 6 reactions

Fast DNA Ladder 1 15 μl

Micropipette tips At least 13

7 wells in an electrophoresis gel

IMPORTANT NOTE: There are several ways to prepare agarose gels.

• Scan the QR code for detailed instructions on how to prepare

Background information P.13

Today’s lab P.19

Laboratory guide 1: Endpoint observation P.22

Laboratory guide 2: qPCR time point observations P.37