Firepol master mix ready to load

Manufactured by Solis BioDyne

Sourced in Estonia

FIREPol® Master Mix Ready to Load is a premixed solution containing all the necessary components for standard PCR reactions. It includes a hot-start Taq DNA polymerase, dNTPs, and reaction buffer.

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Lab products found in correlation

Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Agarose gel, by Merck Group (1 mentions) Axyprep bacterial genomic dna miniprep kit, by Corning (1 mentions) 1 kb dna ladder, by Merck Group (1 mentions) Ethidium bromide, by Bio Basic (1 mentions) Quantity one 4, by Bio-Rad (1 mentions) Firescript rt cdna synthesis kit, by Solis BioDyne (1 mentions) Gel doc xr imaging system, by Bio-Rad (1 mentions) Omniscript rt kit, by Qiagen (1 mentions) Rneasy fibrous tissue mini kit, by Qiagen (1 mentions) Nuclease free water, by Integrated DNA Technologies (1 mentions) Dneasy tissue kit, by Qiagen (1 mentions) Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Ethidium bromide, by Bio-Rad (1 mentions) 1 kb dna ladder, by Solis BioDyne (1 mentions) Gel imaging system, by Bio-Rad (1 mentions) Qiaamp dna stool mini kit, by Qiagen (1 mentions)

Spelling variants of this product

FIREPol Master Mix (45 citations) HOT FIREPol Blend Master Mix Ready to Load (11 citations) 5x Hot FIREPol® Ready to Load Master Mix (2 citations)

13 protocols using firepol master mix ready to load

Listeria monocytogenes Genomic DNA Isolation and PCR

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Total genomic DNA was isolated from L. monocytogenes ATCC 19114 and the listeria isolates using an AxyPrep™ bacterial genomic DNA miniprep kit (Axygen Scientific, Inc., U.S.A.) in accordance with the manufacturer’s instructions. Listeriolysin O gene primer.
For PCR, a 50-μl solution comprising 1× FIREPol® Master Mix Ready to Load (12.5 mM MgCl_2; Solis BioDyne, Tartu, Estonia), 2-μl listeriolysin O gene primer mix (50 pmol), 5-μl DNA template (50 μg/ml), and 33-μl of ultrapure water was used. DNA was amplified in a MULTEGENE thermal cycler (Labnet International, Inc. Edison, NJ) as follows: 95°C, 10 min; followed by 35 sequential cycles of 94°C for 1 min, 52°C for 1 min, 72°C for 1 min; and a final elongation step at 72°C for 10 min was performed after the completion of the cycles. The amplified PCR products, along with a 1-kb DNA ladder (GeneCraft), were separated on a 1.5% agarose gel (Sigma–Aldrich) containing Ethidium Bromide (0.5 mg/ml, ROTH) by electrophoresis (30 min at 100 V in 10× Tris-Borate-EDTA buffer; BIO Basic, Inc.), and visualized using a visual image analyzer software (Syngene).

Yehia H.M., Elkhadragy M.F., Aljahani A.H, & Alarjani K.M. (2020). Prevalence and antibiotic resistance of Listeria monocytogenes in camel meat. Bioscience Reports, 40(6), BSR20201062.

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BOX-PCR Typing of mcr-1-Positive E. coli

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BOX-PCR typing of the mcr-1-positive E. coli was performed using the BOX-A1R primer (5′-CTACGGCAAGGCGACGCTGACG-3′) [36 (link)]. All PCR reactions (25 µL) contained 3 µL of DNA, 0.5 µL Box-A1R, 4 µL master mix (5× FIREPol^® Master Mix Ready to Load, Solis BioDyne, Tartu, Estonia) and 17.5 µL DNase free water. The PCR program was: an initial denaturation step for 2 min at 94 °C followed by 38 cycles, each consisting of 30 s at 94 °C followed by annealing at 50 °C for 1 min and extension for 8 min at 65 °C. The final extension step was for 8 min at 65 °C. PCR products were analyzed on 2% agarose gels containing ethidium bromide. 1 Kb DNA ladder was also loaded in the gel to compare between the BOX-PCR profiles.

Hassan J., Eddine R.Z., Mann D., Li S., Deng X., Saoud I.P, & Kassem I.I. (2020). The Mobile Colistin Resistance Gene, mcr-1.1, Is Carried on IncX4 Plasmids in Multidrug Resistant E. coli Isolated from Rainbow Trout Aquaculture. Microorganisms, 8(11), 1636.

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Semi-Quantitative Analysis of SYNPO2 Isoforms

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For a semi-quantitative analysis of SYNPO2 isoform expression in various human tissues at the RNA level, total RNA was isolated from tissue specimens using the RNeasy Fibrous Tissue Mini Kit following the manufacturer’s instructions (Qiagen, Hilden, Germany). cDNA was prepared using random nonamers and the FIREScript RT cDNA Synthesis Kit (Solis Biodyne, Tartu, Estonia) or the Omniscript RT Kit (Qiagen, Hilden, Germany). cDNA was amplified by PCR using the oligonucleotides listed in Table 1 using FIREPol Master Mix Ready to Load (Solis Biodyne). Oligonucleotides were designed using OligoPerfect Primer Designer (https://www.thermofisher.com/de/de/home/life-science/oligonucleotides-primers-probes-genes/custom-dna-oligos/oligo-design-tools/oligoperfect.html (accessed on 10 January 2023)) by submitting the sequence of the regions flanking the individual exon boundaries. Fragments were analyzed by agarose gel electrophoresis. Gels were photographed and analyzed by densitometry using a GelDoc XR Imaging system and Quantity One 4.6 software (Bio-Rad, Feldkirchen, Germany).

Lohanadan K., Assent M., Linnemann A., Schuld J., Heukamp L.C., Krause K., Vorgerd M., Reimann J., Schänzer A., Kirfel G., Fürst D.O, & Van der Ven P.F. (2023). Synaptopodin-2 Isoforms Have Specific Binding Partners and Display Distinct, Muscle Cell Type-Specific Expression Patterns. Cells, 13(1), 85.

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Molecular Confirmation of Bacterial Identity

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PCR was performed to confirm the identity of the propagated isolates by detecting the β-galactosidase gene using the E. coli-specific lacZ3 primers (Table 1), as previously described [21 (link)]. All PCR reactions were done using a MultiGene Conventional PCR machine (Labnet, Edison, New Jersey, USA). The gene was amplified using 5 μL of PCR Master Mix 5× (FIREPol^® Master Mix Ready to Load, Solis BioDyne, Estonia). The volume was made up to 25 μL using nuclease free water (Integrated DNA Technologies, Coralville, IA, USA). Amplification was done by initial denaturation at 95 °C for 3 min, followed by denaturation at 95 °C for 30 s; annealing temperature of primers was 58 °C for 30 s and extension at 72 °C for 1 min. The final extension was conducted at 72 °C for 10 min and the total reaction was performed for 37 cycles. The amplified PCR products were analysed by electrophoresis in 1.5% agarose gel at 100 v (NanoPAC Power supply, Cleaver Scientific, Rugby, UK) for 45 min, stained with ethidium bromide, and finally, visualized with UV Transilluminator.

Abu-Sini M.K., Maharmah R.A., Abulebdah D.H, & Al-Sabi M.N. (2023). Isolation and Identification of Coliform Bacteria and Multidrug-Resistant Escherichia coli from Water Intended for Drug Compounding in Community Pharmacies in Jordan. Healthcare, 11(3), 299.

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Molecular Identification of Hymenolepididae

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Individual tapeworms (or their parts) were analysed by PCR-derived methods. For this purpose, genomic DNA was isolated by DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer´s instructions. PCR reactions were performed using 5x FIREPol® Master Mix Ready to Load (SOLIS Biodyne, Estonia). The first internal transcribed spacer region (ITS1) with adjacent partial subunits (18S, 5.8S) of ribosomal DNA and the nuclear gene coding for paramyosin (pmy) gave satisfactory DNA profiles in single isolates allowing to derive their relationships with other hymenolepidids using GenBank® deposited sequences. Primers amplifying the ITS1 region (F3, R3) and running conditions were those previously described in Macnish et al. (2002) (link). The final PCR product included 22 bp of the 18S, 587 bp of the ITS1 and 23 bp of the 5.8S (in total 632 bp). For the pmy amplification, a nested PCR approach was employed according to the protocol provided in the above study. Using the external set of primers (Ext-F, Ext-R) a 700 bp product was first amplified. The internal set of primers then amplified DNA template from the primary PCR reaction, resulting in a 617 bp product for H. microstoma. Compositions of primers and running conditions for primary and secondary nested PCRs were those outlined in Macnish et al. (2002) (link), except that 40 cycles were carried out in both reactions.

Jarošová J., Šnábel V., Cavallero S., Chovancová G., Hurníková Z, & Antolová D. (2020). The Mouse Bile Duct Tapeworm, Hymenolepis Microstoma in Free-living Small Mammals in Slovakia: Occurrence and Genetic Analysis. Helminthologia, 57(2), 120-128.

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Apoptotic gene expression in MCF-7 cells

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MCF-7 cells were cultured with different concentrations (10, 20 and 30 µM) of compounds 6 and 10 for 24 h, and the untreated cells were used as a control. The cells were lysed using Trizol reagent (Invitrogen, USA). Total RNA was extracted as per the manufacturer’s instructions. Total RNA was quantitated by taking 2 µL of resuspended RNA on a Nano drop spectrophotometer (Thermo Scientific, USA) and reverse transcribing an equal amount (1 µg) of RNA to make complementary deoxyribonucleic acid (cDNA) using a Super Script VILO cDNA synthesis Kit (Invitrogen), as per the manufacturer’s protocol, in a final volume of 20 µL. The mixture was incubated at 42 °C for 1 h. The generated cDNA (2 µL) was used to assess the mRNA expression of apoptotic genes, including caspase-7, Bax, Bcl-2, and p53. The actin gene was used as an internal control. RT-PCR was performed using 5x Firepol Master Mix ready to load (Solis BioDyne, Tartu, Estonia), as per the manufacturer’s instructions. The specific primer sets used in this study are listed in Table 3. The program was run as follows: initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 45 s, elongation at 72 °C for 45 s (30 cycles), and final extension at 72 °C for 10 min. The RT-PCR products were electrophoresed on a 1.2% agarose gel containing ethidium bromide, and the gel was imaged on a Licor machine.

Alqahtani A.S., Ghorab M.M., Nasr F.A., Ahmed M.Z., Al-Mishari A.A, & Attia S.M. (2021). Novel sulphonamide-bearing methoxyquinazolinone derivatives as anticancer and apoptosis inducers: synthesis, biological evaluation and in silico studies. Journal of Enzyme Inhibition and Medicinal Chemistry, 37(1), 86-99.

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Comprehensive Antibiotic Resistance Profiling

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The bla_TEM, bla_SHV, bla_OXA-1-like and bla_CTX-M (group 1, 2 and 9) genes were detected via M-PCRs, and the bla_CTX-M group 8/25 was detected using a single PCR [50 (link),51 (link)]. The presence of plasmid-mediated AmpC β-lactamase genes, including ACC, FOX, MOX, DHA, CIT and EBC groups, was examined via M-PCR [50 (link)]. The carbapenem-resistant isolates were included for M-PCRs for the detection of carbapenemase-encoding genes [52 (link)]. PMQR genes, including aac(6′)-Ib-cr, qnrA, qnrB, qnrC, qnrD, qnrS and qepA, and a plasmid-mediated azithromycin resistance gene mph(A) were detected using PCRs [53 (link),54 (link),55 (link),56 (link),57 (link),58 (link),59 (link)]. The mcr-1 to -9 genes were screened in the colistin-resistant isolates using M-PCRs [60 (link),61 (link)]. The bla_CTX-M, bla_NDM and mcr variants were identified using nucleotide sequencing. All PCR reactions were carried out in 25 μL of reaction mixture including 5X Firepol^® Master Mix Ready-to-Load (Solis Biodyne, Tartu, Estonia), 0.2 µM of each primer and 1 μL of DNA template. The primers used for the detection of antimicrobial resistance genes are listed in Supplementary Table S2.

Nittayasut N., Yindee J., Boonkham P., Yata T., Suanpairintr N, & Chanchaithong P. (2021). Multiple and High-Risk Clones of Extended-Spectrum Cephalosporin-Resistant and blaNDM-5-Harbouring Uropathogenic Escherichia coli from Cats and Dogs in Thailand. Antibiotics, 10(11), 1374.

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Tomato Variety SSR Marker Evaluation

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A set of ten SSR primers provided by Kaneka Eurogentec, Belgium was selected to be tested with the four tomato varieties: SSR9, SSR20, SSR74, SSR241, SSRX90770, SSR T7, SSR T57, SSR T62, SSR T70, SSR T107. For PCR reactions were used: 5µl of 5x FirePol Master Mix Ready to load (Solis BioDyne, Estonia), 3 µl DNA template, 2 µl of forward and 2 µl reverse primer, and water to a total volume of 25 µl. In a Techne TC-512 Therman Cycler were carried out the cycling amplifications as following: one cycle for initial denaturation 4 min at 94 °C; 35 cycles with 1 min at 94 °C, 1 min at 55 °C, or 58 °C for primers annealing, and 2 min at 72 °C for elongation. The final extension of 7 min at 72 °C, and then maintained at 4 °C.
The amplification products with RAPD and SSR markers were visualized after horizontal electrophoresis in 2.0% or 3.0% agarose gel respectively, with TAE buffer, and stained with ethidium bromide. The gels were photographed with Gene Flash Syngene Bio Imaging system under UV light. GeneRuler of 100 bp DNA Ladder (Solis BioDyne) was used as a molecular weight marker.

Bădulescu A., Popescu C., DUMITRU A.M., & Sumedrea D. (2020). New varieties of tomato - morphological aspects and molecular characterisation with RAPD and SSR markers. Notulae Scientia Biologicae, 12(4), 818-828.

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Comprehensive Molecular Characterization of E. coli

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DNA was extracted from pure bacterial cultures by boiling for 15 min at 95 °C. PCR reactions were then prepared as follows: 3 µL of DNA, 0.5 µL of each of the forward and reverse specific primers, 4 µL of master mix (5× FIREPol^® Master Mix Ready to Load, Solis BioDyne, Tartu, Estonia), and 12 µL of DNase free water. The identity of the E. coli isolates was further confirmed by targeting a species-specific 16S rRNA gene fragment. The PCR analysis also included screening for mcr-1 and other mcr genes (mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, mcr-8) (Table 1). Reactions without DNA were used as a negative control, while DNA from a previously confirmed mcr-1-positive E. coli was used as a positive control [35 (link)]. The amplified products were analyzed by electrophoresis in 1% agarose gel containing ethidium bromide (BioRad, Hercules, CA, USA) and visualized using a gel imaging system (BioRad, Hercules, CA, USA). 1 Kb DNA ladder (Solis BioDyne, Tartu, Estonia) was also loaded in the gel to determine the size of the amplicons.
Using PCR analysis, the E. coli isolates were also screened for other genes, including bla_TEM-1, bla_CTX-M, bla_SHV-1, bla_NDM-1, bla_OXA-48, bla_IMP, bla_KPC, int1, Class 1 Integron gene, Class 2 Integron gene, tetA, tetB, tetC, tetD, tetE, tetG, and strA (Table 1).

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Genetic detection of Echinococcus spp. in fecal samples

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Twenty faecal samples positive for taeniid eggs were analysed by PCR-derived methods. To disrupt the parasite eggshells, faecal samples were homogenized in a Qiagen TissueLyser 85210 (Retsch, Haan, Germany) using 5 mm stainless steel beads for 6 min (30 Hz). After this step, the genomic DNA was isolated using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PCR reactions were performed using the 5× FIREPol^® Master Mix Ready to Load (SOLIS Biodyne, Tartu, Estonia). For detection of Echinococcus spp. tapeworms, a nested PCR reaction was used. Amplification of the partial 12S rRNA gene was performed using specific primers; for the first step primers, P60-for and P375-rev designed by Dinkel et al. [17 (link)] were used. To detect E. multilocularis, the second step was performed with the Em-nest-for and Em-nest-rev primers designed by Dyachenko et al. [18 (link)]. For E. granulosus sensu stricto (s.s.), the primer pair E.g.ss1for and E.g.ss1rev, and for E. canadensis, the primers E.g.cs1for and E.g.cs1rev were used [19 (link)]. To detect other taeniid species, amplification of a 471 bp region of the nad1 gene was applied with the JB3 and JB4.5 primer set, as described by Bowles and McManus [20 (link)].

Jarošová J., Antolová D., Lukáč B, & Maďari A. (2021). A Survey of Intestinal Helminths of Dogs in Slovakia with an Emphasis on Zoonotic Species. Animals : an Open Access Journal from MDPI, 11(10), 3000.

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