qPCR assay for Aquarickettsia spp.
Stephanie Rosales, Sterling R Butler
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
qPCR for the quantification of Aquarickettsia spp. ( Klinges et al., 2022) a putative parasite found in the coral A. cervicornis. This protocol has been altered by incorporating a recently published A. cervicornis CAM control gene (Palacio-Castro et al., 2021) targeted to detect differences across A. cervicornis genotypes because it is a single-copy gene in A. cervicornis .
Steps
Prepare for qPCR
Remove PCR reagents from freezer and allow reagents to thaw on ice or at room temperature.* Wipe down PCR hood with 10% bleach and ethanol.
- Place consumables such as tubes, plates, plate sealers, and water in PCR hood and turn on UV light for
0h 20m 0s - Once everything is thawed vortex PCR reagents, spin them down, and place them on ice.
- Keep reagents cool or on ice during the duration of the protocol.
Prepare PCR master mix
Prepare enough master mix for the number of reactions needed. Each combination of sample and target (gene) should be run at least in duplicates. Add a few reactions to your calculations to account for pipetting errors.
A. cervicornis A. cervicornis (CAM) master mix
| A | B | C |
|---|---|---|
| Component | Volume per Rxn | x rxn + 10% |
| PCR water | 2.4 uL | |
| PrimeTime MM | 5 uL | |
| Forward primer (10 uM) | 0.2 uL | |
| Reverse primer (10 uM) | 0.2 uL | |
| Probe (10 uM) | 0.2 uL | |
| Total MM volume per reaction | 8 uL |
A. rohwerii (tlc1) master mix
| A | B | C |
|---|---|---|
| Component | Volume per Rxn | x rxn + 10% |
| PCR water | 2.4 uL | |
| PrimeTime MM | 5 uL | |
| Forward primer (10 uM) | 0.2 uL | |
| Reverse primer (10 uM) | 0.2 uL | |
| Probe (10 uM) | 0.2 uL | |
| Total MM volume per reaction | 8 uL |
- Combine all the PCR master-mix reagents in a microcentrifuge tube
- Mix gently and spin down to collect mixture and remove bubbles
Setup the qPCR plate
Add 8 uL of master mix to each well. Aiming for the bottom of the well will help to visualize what wells had master mix and DNA added. * Add DNA to each well (2 uL). Aiming for the top of the well will help to visualize what wells had master mix and DNA added.
- Close the plate with optical clear caps or seals
- Spin down the plate to mix the DNA and mastermix.
- Place in the qPCR machine and start the machine using the specified settings.
qPCR thermocyler program settings
Select SYBER green and long run
| A | B | C | D |
|---|---|---|---|
| Procedure | Temperature | Time | Cycle |
| Initial denaturation | 95 C | 3 min | 1 |
| Denaturation | 95 C | 15 sec | 40 |
| Annealing | 60 C | 1 min | 40 |
| Extension | 72 C | 30 sec | 40 |
