To detect the 382-nucleotide deletion in the SARS-CoV-2 genome, we used two specific PCR primers (forward 5ʹ-TGTTAGAGGTACAACAGTACTTT-3ʹ; reverse 5ʹ-GGTAGTAGAAATACCATCTTGGA-3ʹ) flanking the deleted region.5 (link) For samples with high cycle threshold (Ct) values, hemi-nested PCR was done with a second forward primer (5ʹ-TGTTTATAACACTTTGCTTCACA-3ʹ) and the same reverse primer as before. The PCR mixture contained the cDNA primers (10 μM each), 10 × Pfu reaction buffer (Promega, Madison, WI, USA), Pfu DNA polymerase (Promega), and 10 mM dNTP mix (Thermo Scientific, Waltham, MA, USA). PCR was done in a thermal cycler (Applied Biosystems Veriti, Foster City, CA, USA) with the following conditions: 95°C for 2 min, followed by 35 cycles at 95°C for 1 min, 52°C for 30 sec, and 72°C for 1 min; and a final extension at 72°C for 10 min. Deletions in the PCR products were visualised with use of a QIAxcel DNA screening cartridge on QIAxcel Advanced capillary electrophoresis system (Qiagen, Hilden, Germany).
Qiaxcel dna screening cartridge
Manufactured by Qiagen
Sourced in Germany
The QIAxcel DNA screening cartridge is a lab equipment product from Qiagen that is used for the automated analysis of DNA samples. It provides a rapid and efficient method for the separation and detection of DNA fragments, enabling the assessment of DNA quality and quantity.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Dntp mix, by Thermo Fisher Scientific (1 mentions)
Veriti, by Thermo Fisher Scientific (1 mentions)
Pfu dna polymerase, by Promega (1 mentions)
Qiaxcel advanced capillary electrophoresis system, by Qiagen (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Qx alignment marker 15 bp 1 kb, by Qiagen (1 mentions)
1 kb ladder, by Thermo Fisher Scientific (1 mentions)
Gelred, by Biotium (1 mentions)
Qiaxcel advanced system, by Qiagen (1 mentions)
4 protocols using qiaxcel dna screening cartridge
1
PCR Detection of SARS-CoV-2 Genome Deletion
2
LAMP-based Detection of Fungal DNA in Environmental Samples
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LAMP reactions were performed using the OmniAmpTM RNA & DNA LAMP kit (Lucigen Corporation, Middleton, WI) following the manufacturer’s protocol. Briefly, each 12.5 μl reaction consisted of 1 μl of gDNA, 1.25 μl of 10× DNA Polymerase Buffer C, 0.4 μl of 25 mM dNTPs, 1 μl of 100 mM MgSO4, 0.4 μl of 5M betaine, 0.25 μl of OmniAmp DNA Polymerase and 1.25 μl of 100 μM primer mix. The LAMP primer mix consisted of 2 μM each of the F3 and B3 primers, 8 μM each of the LF and LB primers, and 16 μM each of the FIP and BIP primers. Reactions were incubated as recommended by the manufacturer at 70 °C for 30 min in a C1000 Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA). LAMP reactions were performed with templates of 1 ng of fungal gDNA and 10 ng of total DNA isolated from boxwood environmental samples. DNA from C. pseudonaviculata CBS 139707, which was used as a genome reference for the design of primers, served as a positive control. Amplifications were assessed on a QIAxcel capillary gel electrophoresis instrument system (QIAGEN, Germantown, MD) using the QIAxcel DNA screening cartridge (QIAGEN). Reactions were performed a minimum of two times for all samples tested.
3
Rapid Tetracycline Resistance Detection
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Isolates that were phenotypically resistant to tetracycline were tested for tetA and tetB genes. DNA extraction was performed by suspending a single colony from a blood agar plate in 500 μL of deionized water in 1.5 mL microcentrifuge tube, boiling for 10 min at 100°C, and centrifuging at 10,000 x g for 5 min. A duplex PCR assay for tetA and tetB was performed as described by Harvey et al. [25 (link)] with E. coli ATCC 47042 (positive for tetB) and XL1-Blue E. coli strain (positive for tetA) as positive controls. The DNA in 96-well plates were amplified using an Eppendorf Mastercycler gradient thermal cycler (USA Scientific, Inc., Ocala, FL), and PCR products were then transferred to the Automated QIAxcel System (QIAgen, Valencia, CA). Microcapillary electrophoresis was performed using a QIAxcel DNA screening cartridge (QIAgen), a QX alignment marker (15bp/1 kb; QIAgen), and a 50 to 800 bp QX size marker (QIAgen). The electrophoresis was documented and analyzed for the presence of specific bands.
4
Assessing PCR Amplicon Quality and Quantification
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PCR products for all negative and positive controls were electrophoresed at 80 V on a 1% agarose gel for 30 min in tris-acetate-ethylenediamine tetraacetic acid (EDTA) buffer (TAE; 40 mM Tris, 20 mM acetic acid, 1 mM EDTA); stained with GelRedTM (10X, Biotium, Fremont, CA); and run alongside a 1-kb ladder (Thermo Fisher Scientific, Grand Island, NY). Gels were visualized using an UltraCam Digital Imaging System (BioRad, Hercules, CA). Amplicon quality was assessed using the QIAxcel DNA Screening cartridge (Qiagen). Briefly, 2 μL PCR product generated in the second amplification was added to 8 μL QX DNA dilution buffer and visualized using high-resolution capillary electrophoresis on a QIAxcel Advanced System (Qiagen). Samples with a peak at the appropriate amplicon size and minimum levels of primer dimers were deemed as having adequate quality. DNA (2 μL amplicon combined with 198 μL of a solution Qubit dsDNA HS reagent and Qubit HS buffer in a 1:200 dilution) was quantified using the Qubit® 2.0 Fluorometer and the QubitTM dsDNA High Sensitivity Assay (Thermo Fisher Scientific, Waltham, MA). Samples were pooled to contain 50 ng DNA from each sample. If samples amplified poorly, an attempt at re-amplification was made. Subsequently, amplicons were combined if necessary to obtain sufficient amounts of DNA, or the entire volume from the PCR was used.
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