Brewery QC - PCR Protocols and Primers (STA1, and more)
PCR Primers and Protocols
We happily supply the PCR primers used for quality control and research in-house at Escarpment Labs.
The STA1/ITS protocol is the same. I tweaked it so we would able to run the STA1 primers for our experiments while using the ITS primers as a control to confirm PCR amplification worked as intended.
And the STA1 primer sequences are:
DNA extraction for both of these protocols follows the Instagene Matrix protocols as instructed. The PCR protocol is:
- 94°C for 15 minutes
- 30 cycles of denaturing at 94°C for 1 minute, annealing at 49°C for 2 minutes, and extension at 72°C for 2 minutes
- 72°C for 10 minutes,
- 4°C hold for infinity.
More information can be found on this blog: https://www.escarpmentlabs.com/single-post/2018/10/16/Demystifying-diastaticus-part-1
V6r and V3kl (we just call it the V6r PCR) are the primers we use to amplify the 16s rRNA gene found in all bacteria. The paper we sourced them from, which includes the primers, can be found here: https://www.researchgate.net/publication/330725309_Effects_of_defined_gut_microbial_ecosystem_components_on_virulence_determinants_of_Clostridioides_difficile
Our working protocol involves using bead tubes commercially available through Sigma (BeadBug 2mL profiled tubes - Z763721-50EA) and Instagene matrix from Biorad. Prior to Instagene Matrix, place a colony into 1mL of sdH2O. Mix vigorously with a pipette until the colony is fully suspended in water. Vortex the sample for 1 min using the Vortex Genie. Transfer the entire contents to a pre-filled bead tube. Vortex the samples for 2-3 minutes using the Vortex Genie. Spin down the samples at 10,000rpm for 2 minutes. Transfer 30µL of the supernatant to 200µL of the Instagene Matrix (BioRad), vortex 1 min with Vortex Genie, and follow the Instagene protocol as suggested (30 min bath at 56C, 9 min boil at 100C).
The PCR Protocol is:
- 94°C for 2 min;
- 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds; x30 cycles;
- 72°C for 5 mins
- 4°C hold for infinity
For both the ITS and V6r, we clean up the PCR product once confirming amplification worked via gel electrophoresis using a Qiagen kit, and submit it to a third party for sequence ID. Once those results are returned to us, we BLAST the results to determine the species identity. (I can explain this process to you later if you need me to).