Strains were grown in 10 mL of LB broth at 37°C for 18–24 h up to the late exponential phase and collected by centrifugation. Total RNA extraction was carried out using the QIAGEN RNeasy purification kit. After checking the RNA extraction quality on a 1% agarose gel and measuring the RNA content (Nanodrop, Thermo Fisher Scientific, France), RNA extracts were stored at −20°C until further use. Prior to cDNA synthesis, genomic DNA (gDNA) was removed from 1 μg of total RNA using the gDNA wipeout buffer included in the QuantiTect Reverse Transcription kit (QIAGEN). The reverse transcription was performed in a volume of 20 μL including 14 μL of template RNA (extract concentrations adjusted to contain 1 μg of RNA), 1 μL of Reverse Transcription Master Mix, 4 μL of RT buffer 5× (containing dNTPs and Mg2+) and 1 μL of RT primer mix. Reverse transcription was performed in a Veriti PCR Thermal Cycler (Applied Biosystems, France) for 30 min at 42°C followed by a 3 min incubation at 95°C to inactivate the reverse transcriptase. All reactions including RNA handling were carried out on ice. The rpsL gene was used as reference to normalize the relative amount of mRNA.7 (link)
Veriti pcr thermal cycler
Manufactured by Thermo Fisher Scientific
Sourced in France
The Veriti PCR Thermal Cycler is a laboratory instrument used for the amplification of DNA samples through the process of Polymerase Chain Reaction (PCR). The device precisely controls the temperature and duration of the various stages required for DNA replication, enabling the efficient and reliable production of targeted DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Rneasy purification kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
3500xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
4 protocols using veriti pcr thermal cycler
1
RNA Extraction and Reverse Transcription
2
Sequencing CHIKV Nonstructural Genes Across Pakistan
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Eight CHIKV isolates (two from each province) were randomly selected for complete CHIKV nonstructural protein coding nucleotide sequences from different geographic regions namely Sindh, Baluchistan KPK and Punjab.
Specific CHIKV oligonucleotide primers [4 (link)] were used for RT-PCR/sequencing of complete nonstructural genes (NsP1, NsP2, NsP3, and NsP4) from prototype African strain S-27 as summarized inS1 Table . The reverse transcription RT-PCR assay was carried out using Qaigen one Step RT-PCR kit (GmbH) in a total 50μl volume with 20 μmol each of forward and reverse primers and 3 μl of the extracted RNA [19 (link)]. PCR reactions were conducted on a Veriti PCR thermal cycler (Applied Biosystems, USA) using the protocol; 30 min at 42°C for reverse transcription and 3 min at 94°C for Taq polymerase activation; 35 cycles of for 45 sec at 94°C, 1 min at 54°C, 1 min at 60°C and final extension step for 5 min at 60°C. Amplified products were visualized on 1.5% agarose gel.
Specific CHIKV oligonucleotide primers [4 (link)] were used for RT-PCR/sequencing of complete nonstructural genes (NsP1, NsP2, NsP3, and NsP4) from prototype African strain S-27 as summarized in
3
Sequencing and Analysis of Antibiotic Resistance Genes in Pseudomonas aeruginosa
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The mucA gene of all PA and for quinolone resistant QRDR genes (gyrA, gyrB, parC and parE) for 43 PA isolates included in this study were amplified by PCR. Primers used for amplification and sequencing are reported in Table 1
4
Hotspot Mutation Analysis in Oncogenes
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The analysis of hot spot mutations of TERT promoter regions (containing the sites c.-124:C>T and c.-146:C>T), KIT (exons 9, 11, 13, and 17), PDGFRA (12, 14, and 18), BRAF (exon 15), and NRAS (codons 12/13 and 61) was performed by PCR followed by direct Sanger sequencing, as described previously by our group.22 (link),23 (link) Briefly, using specific pairs of primers (Appendix Table A1 ), the target regions were amplified by PCR using a Veriti PCR thermal cycler (Applied Biosystems, Foster City, CA). We used an initial denaturation at 95°C for 15 minutes followed by 40 cycles of 95°C denaturation for 45 seconds; specific annealing temperature was for 90 seconds and the 72°C elongation phase for 45 seconds followed by a 72°C final elongation for 7 minutes. Amplification of PCR products was confirmed by gel electrophoresis.
Sequencing PCR was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3500xL Genetic Analyzer (Applied Biosystems). Confirmation of all mutations was performed by repeating the PCR and sequencing the altered regions.
Sequencing PCR was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3500xL Genetic Analyzer (Applied Biosystems). Confirmation of all mutations was performed by repeating the PCR and sequencing the altered regions.
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