2-(4-(Dimethylamino)phenyl)-3,6-dimethylbenzo[d]thiazol-3-ium chloride (ThT) dye was purchased from Sigma-Aldrich (Gillingham, UK) and used without further purification. The stock solution (5 mM) was prepared in DNase- and RNase-free water; aliquots were stored at −20°C. Fifty microliters of INS-intron-1-derived oligonucleotides and controls diluted to 20 μM were heated at 90°C for 5 minutes and cooled down to room temperature for 1 h at a rate of 1°C/min on a PeqSTAR 96 Universal Gradient thermocycler (PeqLab, Fareham, UK). ThT was added to a final concentration of 80 μM, which was derived by assay optimizations. ThT fluorescence intensities were measured at 508 nm (pH 7.2) using the FLx800 Microplate Fluorescence Reader (Biotek Instruments, Swindon, UK). The assay sensitivity was set to 90. INS-derived oligonucleotides and negative and positive controls were prepared and measured three times. The average 10 technical replicates were measured for each sample preparation. Positive controls included c-myc derived from the promoter sequence of the c-myc proto-oncogene, Plas24 derived from a plasmodium telomere and 21DNA from human DBF4B (de la Faverie et al.21 (link) and references therein).
Peqstar 96 universal gradient thermocycler
Manufactured by Avantor
Sourced in United Kingdom, Germany
The PeqSTAR 96 Universal Gradient thermocycler is a laboratory instrument designed for DNA amplification. It features a 96-well block that can accommodate various sample formats and supports temperature gradient functionality for optimization of PCR reactions.
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Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Flx800 fluorescence microplate reader, by Agilent Technologies (1 mentions)
Gel doc 1000 system, by Bio-Rad (1 mentions)
Peqgold trifast, by Avantor (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Dreamtaq green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Gelred, by Biotium (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
Brilliant 3 ultra fast sybr green qpcr master mix, by Agilent Technologies (1 mentions)
Rotor gene q thermocycler, by Qiagen (1 mentions)
5 protocols using peqstar 96 universal gradient thermocycler
1
Thioflavin T Fluorescence Assay for INS-derived Oligonucleotides
2
Fluorescence Measurement of G4-Binding NMM
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NMM (Frontier Scientific, Logan, UT, USA) was dissolved in DNase- and RNase-free water and kept at 4°C at a stock concentration of 100 μM. N-methyl mesoporphyrin IX (NMM) was added to oligonucleotides to a final concentration of 25 μM. Prior to NMM addition, G4 formation was promoted by heating the samples at 90°C for 5 minutes and cooling down to room temperature on a PeqSTAR 96 Universal Gradient thermocycler (PeqLab, Fareham, UK) for 1 h at a rate of 1°C/min. NMM fluorescence intensity was measured with a Varioskan multireader (Thermo Fisher Scientific, Waltham, MA, USA). Excitation wavelength was at 400 nm. Spectra were collected for a range between 435 and 740 nm.
3
Isolation and Quantification of Total RNA
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Total RNA was isolated using peqGOLD TriFast™ (PEQLAB, Erlangen, Germany). This method is based on the single step RNA isolation described by Chomczynski and Sacchi [34 (link)]. After quantification, 2 μg total RNA were used for preparing DNA-free RNA through desoxyribonuclease I digestion (DNase I, Thermo Fisher Scientific) and a reverse transcription PCR was performed by applying the High Capacity© cDNA archive kit (Applied Biosystems Inc., Foster City, CA, USA), and both steps were carried out according to the manufacturers' protocol.
The housekeeping gene β-actin was amplified for all performed PCRs as control (Figure 2 ). The reactions consisted of 1 × DreamTaq™ Green PCR Master Mix (Thermo Fisher Scientific), 1 and, respectively, 2 µl cDNA (for ß-actin and, resp., PRL PCR) cDNA from the reverse transcription, 0.3 μM forward and reverse primer, and dH2O ad 25 μL. The PCRs were carried out in a peqSTAR 96 universal gradient thermocycler (PEQLAB). The according primer sequences are shown in Table 1 . PCR products were immediately separated electrophoretically in a 1% agarose gel in presence of the nucleic acid gel stain GelRed™ (1 : 10.000, Biotium, Hayward, USA) or stored at −20°C for subsequent electrophoresis. After separation the agarose gel was analyzed by the GelDoc 1000 system (Bio-Rad Laboratories, Hercules, USA).
The housekeeping gene β-actin was amplified for all performed PCRs as control (
4
Real-time and Semiquantitative PCR Protocols
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All quantitative real-time PCRs (qPCR) were realized in a Rotor-Gene Q thermocycler (Qiagen, Venlo, The Netherlands) using the following program (180 s at 95 °C followed by 40 cycles of 15 s at 95 °C; TA 40 s) and Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA). Quantification of gene expression was calculated using the 2−ΔΔct-method [51 (link)]. Primers designed for quantitative real-time PCR are listed in a data supplement (Supplementary Table S1 ). Semiquantitative PCR was carried out in a peqSTAR 96 Universal Gradient thermocycler (Peqlab, Erlangen, Germany) using either primers shown in Supplementary Table S1 and a mRNA template isolated from adult leaves and seedlings, or primers shown in Supplementary Table S2 and an mRNA template extracted from adult leaves after cold treatment. The PCR program was designed as follows: 1 × 95 °C for 180 s, 40 cycles, 95 °C for 20 s, 50 °C for 30 s, 68 °C for 60 s, 1 × 68 °C for 600 s. Taq DNA Polymerase (New England Biolabs, Ipswich, MA, USA) was used for semiquantitative PCR. PCR products were separated on 1% agarose gels.
5
PCR Amplification of Shiga Toxin-Producing E. coli
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Only isolates of O26, O103, O111, and O157 serogroups (n = 171) were evaluated by LAB B due to time and labor constraints. The original DNA template extracted using NucleoSpin Tissue kits by LAB A was used for approximately 50% of isolates from each serogroup, while for the remaining half, glycerol stocks were re-grown a second time and DNA was extracted using NucleoSpin kits. Both sources of DNA were used as insufficient original extracted DNA was available to complete all LAB B studies. Briefly, 1 µL of DNA template was added to 24 µL PCR mix containing 2× PyroMark Master Mix (Qiagen) and primers as described by Goji et al. [11 (link)]. PCR amplification was performed using a peqSTAR 96 Universal Gradient thermocycler (PeqLab, Erlangen, Germany), with the hotstart program starting at 95 °C for 15 min, 45 cycles at 94, 56 and 72 °C for 30 s each and final extension at 72 °C for 5 min.
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