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Dreamtaq hot start pcr master mix

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DreamTaq Hot Start PCR master mix is a pre-mixed solution containing all the necessary components for performing polymerase chain reaction (PCR) amplification, including the DreamTaq DNA polymerase, dNTPs, and buffer. The Hot Start feature ensures that the DNA polymerase remains inactive until the initial denaturation step, reducing the formation of non-specific amplification products.

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Lab products found in correlation

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13 protocols using dreamtaq hot start pcr master mix

1

Evaluating PCR Bias in Amplifying L1 Loci

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To illustrate the PCR bias when amplifying the pre- and post-integration sites together, we tested amplification on a concentration gradient of a known L1 template extracted from the reporter plasmid GL1#1, including 248bp upstream, 449bp L1#1 and 429bp downstream sequence. We added 1×10−4ng, 1.43×10−5ng, 2.04×10−6ng, 2.92×10−7ng, and 4.16×10−8ng of the L1#1 template (1126bp) to 22.8ng NA12878 genomic DNA to make allele frequency of L1#1 at 92.4%, 64.6%, 20.7%, 3.59% and 0.53%, respectively. We then tested PCR amplification with external primers in the flanking sequences, using PhusionTaq or DreamTaq polymerases, and 30 or 60 PCR cycles (Extended Data Fig. 5c). The PhusionTaq PCR reactions were incubated in a volume of 20 μl, containing 10 μl Phusion green Hotstart II HF PCR master mix (2×, Thermo Fisher), 0.9 μM of the primers, and the relevant template DNA. The primer and L1#1 template sequences are in Supplementary Table 6. The reactions were incubated as follows:
Similarly, the DreamTaq PCR reactions were incubated in a volume of 20 μl, containing 10 μl DreamTaq Hot Start PCR master mix (2×, Thermo Fisher), 0.9 μM of the primers, and the relevant template DNA. The reactions were incubated as follows:
Zhu X., Zhou B., Pattni R., Gleason K., Tan C., Kalinowski A., Sloan S., Fiston-Lavier A.S., Mariani J., Petrov D., Barres B.A., Duncan L., Abyzov A., Vogel H., Moran J.V., Vaccarino F.M., Tamminga C.A., Levinson D.F, & Urban A.E. (2021). Machine learning reveals bilateral distribution of somatic L1 insertions in human neurons and glia. Nature neuroscience, 24(2), 186-196.
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2

Escherichia coli Phylogenetic Grouping

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Escherichia coli phylogenetic groups (PG) were determined as described by Clermont and colleagues (2013 (link)) by quadruplex PCR amplification of three genes (arpA, chuA and yjaA) and a DNA fragment (TspE4.C2) using custom-synthesized primers (Microsynth, Balgach, Switzerland) and a DreamTaq hot start PCR master mix (Thermo Fisher Scientific, Waltham, USA). PCR conditions were as described by Clermont and colleagues (2013 (link)). Bands were visualized with GelRed (Biotium Inc., Fremont, USA) on a TBE gel (2% agarose, 35 min, 100 V), and strains with ambiguous band patterns were subjected to confirmatory C- or E-PCR.
Gekenidis M.T., Schöner U., von Ah U., Schmelcher M., Walsh F, & Drissner D. (2018). Tracing back multidrug-resistant bacteria in fresh herb production: from chive to source through the irrigation water chain. FEMS Microbiology Ecology, 94(11), fiy149.
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3

Phylogenetic Profiling of E. coli Strains

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E. coli phylogenetic groups (PG) were determined as described by Clermont and colleagues [60 (link)] by quadruplex PCR amplification. PCR was performed with custom-synthesized primers (Microsynth, Balgach, Switzerland) and a DreamTaq hot start PCR master mix (Thermo Fisher Scientific, Waltham, USA). Upon band visualization on a TBE gel (2% agarose, 35 min, 100 V), strains displaying ambiguous patterns were subjected to confirmatory C- or E-PCR.
Gekenidis M.T., Qi W., Hummerjohann J., Zbinden R., Walsh F, & Drissner D. (2018). Antibiotic-resistant indicator bacteria in irrigation water: High prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. PLoS ONE, 13(11), e0207857.
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4

Total RNA Extraction and RT-PCR

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Total RNA was extracted using TRIZOL (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Reverse transcription was carried out using Superscript IV (ThermoFisher Scientific, Waltham, MA). Briefly, 2 µg of RNA was converted to cDNA at 65 °C for 5 min followed by a 1 min incubation on ice, 23 °C for 10 min, 50 °C for 10 min and 80 °C for 10 min. PCR primers and annealing temperatures are listed in Supplementary Table S3. The PCR reaction was carried out with DreamTaq Hot Start PCR Master Mix (ThermoFisher Scientific, Waltham, MA) at 95 °C for 3 min, 95 °C for 30 s, followed by 30 cycles at the annealing temperature for 30 s and 72 °C for 1 min.
Yan J., Mehta S., Patel K., Dhupar N., Little N, & Ong Tone S. (2024). Transcription factor 4 promotes increased corneal endothelial cellular migration by altering microtubules in Fuchs endothelial corneal dystrophy. Scientific Reports, 14, 10276.
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5

Polymerase Chain Reaction Amplification

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Polymerase chain reaction (PCR) was performed in a 20 µL reaction containing 8 ng of DNA, 0.2 µM of each primer, DreamTaq™ Hot Start PCR Master Mix (ThermoFisher Scientific Waltham, MA, USA) containing Taq polymerase, and all other reagents. PCR amplification was performed in a Primus 96+ thermocycler (MWG-Biotech®, Luxembourg, Luxembourg), and amplicons were analyzed as described by Luro et al. (2008) with the ten pairs of primers used by Ferrer et al. (2021) [61 (link),62 (link)].
Ferrer V., Paymal N., Costantino G., Paoli M., Quinton C., Tomi F, & Luro F. (2023). Correspondence between the Compositional and Aromatic Diversity of Leaf and Fruit Essential Oils and the Pomological Diversity of 43 Sweet Oranges (Citrus x aurantium var sinensis L.). Plants, 12(5), 990.
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6

SARS-CoV-2 Envelope Gene In Vitro RNA Transcript

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An in vitro RNA transcript of the SARS-CoV-2 envelope gene was generated as a standard for rRT-PCR quantitative detection in human specimens. Viral RNA from a positive clinical sample was used as an initial template for in vitro RNA transcription. cDNA was synthetized using SuperScript™ III First-Strand Synthesis System and random hexamers primer (Thermo Fisher, USA). Double-stranded DNA containing the 5′-T7 RNA polymerase promoter sequence for the SARS-CoV-2 complete E gene sequence, was obtained using DreamTaq Hot Start PCR Master Mix (Thermo fisher, USA) and E-Std-T7-Fwd (TAA TAC GAC TCA CTA TAG GGG CGT GCC TTT GTA AGC ACA A), and the E-Std-Rev (GGC AGG TCC TTG ATG TCA CA) primers [41 (link)]. The DNA was finally transcribed using the MEGAscript T7 Transcription Kit (Thermo Fisher Scientific). The RNA transcripts were purified with Ampure XP beads (Belckman Counter, USA) and quantified with a Qubit fluorometer using a Qubit RNA HS Assay Kit (Thermo Fisher Scientific). All commercial reagents were used according to manufacturer instructions.
Vesga O., Agudelo M., Valencia-Jaramillo A.F., Mira-Montoya A., Ossa-Ospina F., Ocampo E., Čiuoderis K., Pérez L., Cardona A., Aguilar Y., Agudelo Y., Hernández-Ortiz J.P, & Osorio J.E. (2021). Highly sensitive scent-detection of COVID-19 patients in vivo by trained dogs. PLoS ONE, 16(9), e0257474.
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7

Evaluating PCR Bias in Amplifying L1 Loci

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To illustrate the PCR bias when amplifying the pre- and post-integration sites together, we tested amplification on a concentration gradient of a known L1 template extracted from the reporter plasmid GL1#1, including 248bp upstream, 449bp L1#1 and 429bp downstream sequence. We added 1×10−4ng, 1.43×10−5ng, 2.04×10−6ng, 2.92×10−7ng, and 4.16×10−8ng of the L1#1 template (1126bp) to 22.8ng NA12878 genomic DNA to make allele frequency of L1#1 at 92.4%, 64.6%, 20.7%, 3.59% and 0.53%, respectively. We then tested PCR amplification with external primers in the flanking sequences, using PhusionTaq or DreamTaq polymerases, and 30 or 60 PCR cycles (Extended Data Fig. 5c). The PhusionTaq PCR reactions were incubated in a volume of 20 μl, containing 10 μl Phusion green Hotstart II HF PCR master mix (2×, Thermo Fisher), 0.9 μM of the primers, and the relevant template DNA. The primer and L1#1 template sequences are in Supplementary Table 6. The reactions were incubated as follows:
Similarly, the DreamTaq PCR reactions were incubated in a volume of 20 μl, containing 10 μl DreamTaq Hot Start PCR master mix (2×, Thermo Fisher), 0.9 μM of the primers, and the relevant template DNA. The reactions were incubated as follows:
Zhu X., Zhou B., Pattni R., Gleason K., Tan C., Kalinowski A., Sloan S., Fiston-Lavier A.S., Mariani J., Petrov D., Barres B.A., Duncan L., Abyzov A., Vogel H., Moran J.V., Vaccarino F.M., Tamminga C.A., Levinson D.F, & Urban A.E. (2021). Machine learning reveals bilateral distribution of somatic L1 insertions in human neurons and glia. Nature neuroscience, 24(2), 186-196.
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8

Mitochondrial Cytb gene amplification

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Approximately 460 bp of the mitochondrial Cytb gene was amplified using primers mcb398 and mcb869 [31 (link)], with universal tailed sequences on each primer that are compatible with the ONT PCR Barcoding Expansion kit EXP-PBC001 (ONT, Oxford, UK) (Table S1). These primers were designed from an alignment of 67 animal species, and validated for mammals, reptiles, and birds [31 (link)].
PCR was carried out with 6.25 µL DreamTaq HotStart PCR Master Mix (Thermo Fisher, Waltham, MA, USA), 1.25 µL DNA template, and 2 µL of each primer (10 µM stock) in a final volume of 12.5 µL. Cycling conditions were: 95 °C for 3 min; 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final extension of 72 °C for 5 min. All Chelex extractions were diluted for the DNA Barcoding PCR as described in Text S1. PCR products were purified using 1.8× Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA), tested for purity using the NanoDrop™ One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and quantified fluorometrically using the Qubit dsDNA High sensitivity kit.
Seah A., Lim M.C., McAloose D., Prost S, & Seimon T.A. (2020). MinION-Based DNA Barcoding of Preserved and Non-Invasively Collected Wildlife Samples. Genes, 11(4), 445.
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9

16S rRNA gene amplification protocol

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Amplification of the 16S rRNA gene (V3–V4) was performed using V3 (5′-CCT TACGGGAGGCAGCAG-3′), V4 (5′-GGA CTACHV GGG TWTCTAAT-3′) primers (W: A or T; V: G or C or A; H: A or C or T). The primers were custom synthesized by Integrated DNA Technologies. The PCR mixture for every reaction tube was composed of 12.5 µL DreamTaq™ Hot Start PCR Master Mix (Thermo Fisher Scientific) at a 2× concentration, along with 11.5 µL nuclease-free water and 0.5 µL of each primer (V3 forward primer and V4 reverse primer). 1 µL (10 ng/µL) template DNA was added to the 25 µl mastermix. Thermocycling conditions included an initial denaturation step (3 min at 94 °C), followed by 30 cycles of denaturation (30 s at 94 °C), annealing (30 s at 56 °C), extension (5 min at 72 °C), and a final extension step of 1 min at 72 °C. PCR products were separated using gel electrophoresis on 1.5% agarose gel.
Tan Z., Yang W., O’Brien N.A., Pan X., Ramadan S., Marsh T., Hammer N., Cywes-Bentley C., Vinacur M., Pier G.B., Gildersleeve J.C, & Huang X. (2024). A comprehensive synthetic library of poly-N-acetyl glucosamines enabled vaccine against lethal challenges of Staphylococcus aureus. Nature Communications, 15, 3420.
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10

Mitochondrial Cytb Gene Amplification

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Approximately 460 bp of the mitochondrial Cytb gene was amplified using primers mcb398 and mcb869 (Verma & Singh, 2003) (link), with universal tailed sequences on each primer that are compatible with the ONT PCR Barcoding Expansion kit EXP-PBC001 (ONT, Oxford, UK) (Table S1). These primers were designed from an alignment of 67 animal species, and validated for mammals, reptiles and birds (Verma & Singh, 2003) (link).
PCR was carried out with 6.25 µL DreamTaq HotStart PCR Master Mix (Thermo Fisher, Waltham, MA, USA), 1.25 µL DNA template, and 2 µL of each primer (10 µM stock) in a final volume of 12.5 µL. Cycling conditions were: 95°C for 3 minutes; 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds; and a final extension of 72°C for 5 minutes. All Chelex extractions were diluted for the DNA Barcoding PCR as described in Appendix I. PCR products were purified using 1.8X Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA), tested for purity using the NanoDrop™ One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and quantified fluorometrically using the Qubit dsDNA High sensitivity kit.
Seah A., Lim M., McAloose D., Prost S., & Seimon T.A. (2020). MinION-based DNA barcoding of preserved and non-invasively collected wildlife samples.
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