DNA was extracted from the five diatom cultures, following the instructions of the NucleoSpin® Plant II Mini Kit (Macherey and Nagel, Düren, Germany). For identification, the rbcL gene (RuBisCO large subunit) was amplified in a PCR using a commercial PCR-Mastermix (Bioline). The primers Diat-rbcl-iR and Diat-rbcl-F were applied with the respective PCR protocol (Abarca et al., 2014 (link)). Sequencing was carried out by a commercial company (Eurofins, Luxembourg), using the same forward primer as for PCR. The sequences were uploaded to NCBI under the accession numbers OR387857–OR387860.
Pcr master mix
Manufactured by Meridian Bioscience
Sourced in United Kingdom, United States
PCR master mix is a pre-formulated solution containing the necessary reagents for performing polymerase chain reaction (PCR) amplification of DNA. It includes DNA polymerase, nucleotides, and buffer components required for the PCR process.
Automatically generated - may contain errors
4 protocols using pcr master mix
1
Diatom Identification via rbcL Sequencing
2
Extraction and Analysis of Total RNA
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Total RNA was extracted from each leaf tissue sample with a standard TRIzol procedure. The leaf tissues frozen with liquid nitrogen were ground in 800 μl of trizol and incubated at 22 °C for 5 min. After the insoluble pieces were removed by centrifugation, 160 μl of chloroform was added to the supernatant, mixed vigorously for 15 seconds and incubated at 22 °C for 3 min. The two aqueous phases were separated in a centrifuged at 4 °C for 15 min and the upper layer transferred to a new tube was mixed with 500 μl of isopropanol. The sample was incubated at 22 °C for 10 min and centrifuged at 4 °C for 10 min. The pellet was resuspended in 800 μl of 75% ethanol and centrifuged again at 4 °C for 10 min. The pellet was air-dried and re-suspended in 50 μl of molecular biology grade water. The RNA samples were treated with DNase using Ambion DNA-free kit (Life Technologies) and the cDNA for each gene was generated with its corresponding reverse primer using Sensiscript® Reverse Transcription kit (Qiagen) according to the manufacturer’s protocols. The cDNA samples were amplified with the PCR master mix (Bioline) and analyzed in a 1% agarose gel.
3
Detecting Fowl Adenovirus in Embryonic Liver
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The liver homogenates at E2, E5, E10, E15, and E20 were extracted for DNA by using i-Genomic DNA Extraction Mini kit (iNtRON, Korea) according to the manufacturer's recommendations. DNA concentration was measured by biophotometer (Eppendorf, Germany) at 260/280 nm wavelengths. Amplification of DNA for detection of FAdV was conducted by using a PCR master mix (Bioline, UK) according to the manufacturer's protocol. Two set of published primers based on the hexon gene, H1/H2 and H3/H4 [19 (link)], and a newly designed fiber gene primer, FibF/FibR (FibF: 5′-GGTCTA CCCCTTTTGGCTCC-3′ and FibR: 5′-GCGTCGTAGATGA AGGGAGG-3′) based on FAdV-8 reference strain [18 (link)], were used in this study. The PCR product was analyzed via 1% agarose gel electrophoresis at 70 V for 45 min prior to staining with RedSafe Nuclei Acid Staining solution (iNtRON) and visualized under UV transillumination. All positive products were purified by using a MEGAquick-spin Total Fragment DNA Purification kit (iNtRON) following the recommended protocol. The purified product was cloned into pCR 2.1-TOPO vector using a TOPO TA Cloning kit (Invitrogen, USA) prior plasmid purification by DNA-spin plasmid DNA purification kit (iNtRON).
4
PCR Detection of Fosfomycin Resistance Genes
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Conventional PCR was performed to detect fos genes and CTX-M gene in Fosfomycin-resistant isolates using primers and conditions listed in Table 1 [13 , 15 (link)–18 (link)]. The amplification reactions were carried out in 25 µl volumes containing 12.5 µl of PCR master mix (Bioline, USA Inc.), 1 µl of each primer (10 pmol/µl), 8.5µL nuclease-free water, and 2 µl of 100 ng/µl template DNA.
PCR Primers and conditions of the studied genes
| Gene | Primer sequence | Annealing temp | Product size(bp) |
|---|---|---|---|
| arpA | F-AACGCTATTCGCCAGCTTGC R-TCTCCCCATACCGTACGCTA | 59 °C | 400 |
| chuA | F-ATGGTACCGGACGAACCAAC R-TGCCGCCAGTACCAAAGACA | 288 | |
| yjaA | F-CAAACGTGAAGTGTCAGGAG R-AATGCGTTCCTCAACCTGTG | 211 | |
| tspE4C2 | F-CACTATTCGTAAGGTCATCC R-AGTTTATCGCTGCGGGTCGC | 152 | |
| fosA | F-ATCTGTGGGTCTGCCTGTCGT R-ATGCCCGCATAGGGCTTCT-3’ | 59.5 °C | 271 |
| fosA3 | F- CCTGGCATTTTATCAGCAGT R- CGGTTATCTTTCCATACCTCAG | 57.5 °C | 221 |
| fosA4 | F-CTG GCG TTT TAT CAG CGG TT R -CTT CGC TGC GGT TGT CTT T | 60 °C | 234 |
| fosA5 | F-TATTAGCGAAGCCGATTTTGCT R-CCCCTTATACGGCTGCTCG | 55 °C | 177 |
| fosA6 | F- GCTACGGTTCAGCTTCCAGA R- CGAGCGTGGCGTTTTATCAG | 58 °C | 242 |
| fosB | F-ATATGATCAAAGGAATAAATC R-CATATGAAAATTCATATGAGG | 40 °C | 767 |
| fosC2 | F-TGGAGGCTACTTGGATTTG R-AGGCTACCGCTATGGATTT | 50.5 °C | 217 |
| blaCTX−M | F-SCS ATG TGC AGY ACC AGT AA R-CCG CRA TAT GRT TGG TGG TG | 57 °C | 554 |
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