Startwarm hs pcr mix

Manufactured by A&A Biotechnology

Sourced in Poland

StartWarm HS-PCR Mix is a high-sensitivity PCR reagent mix designed for reliable and efficient amplification of target DNA sequences. The mix contains all necessary components for PCR, including a thermostable DNA polymerase, dNTPs, and reaction buffer. This product is suitable for a wide range of PCR applications.

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Lab products found in correlation

Generuler 1 kb dna ladder, by Thermo Fisher Scientific (2 mentions) Midori green advance, by Nippon Genetics (2 mentions) Ethidium bromide, by Merck Group (1 mentions) Surecycler 8800, by Agilent Technologies (1 mentions) Midori green advance dye, by Nippon Genetics (1 mentions) Agarose gel, by Merck Group (1 mentions) Myseq, by Illumina (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions)

7 protocols using startwarm hs pcr mix

Multiplex PCR Screening for Virulence and Carbapenemase Genes

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PCR amplification was used to detect virulence (uge, wabG, and fimH), and carbapenemase (bla_NDM-1) genes. Primers’ sequences are listed in Table 4. Reference strains, including E. coli ATCC 25922, K. pneumoniae ATCC BAA-2473 and K. pneumoniae ATCC 700603, were used as positive controls.
PCR was conducted using StartWarm HS-PCR Mix (A&A Biotechnology, Gdynia, Poland) mixture. Amplification was conducted using the Applied Biosystems Veriti 96 Well Thermal Cycler (Applied Biosystems, Norwalk, CT, USA) with the following protocol: initial denaturation at 95 °C for four min was followed by 35 cycles of amplification (denaturation—95 °C for 30 s, annealing—53 °C and 52 °C for 30 s respectively for uge/wabG/fimH and bla_NDM-1 genes, extension—72 °C for 60 s) and finished with final extension at 72 °C for 10 min. After PCR, obtained products were analysed by electrophoresis (60 min, 100 V, 1 × tris/borate/ethylenediaminetetraacetic acid) in agarose gel (1.5%, w/v; DNA Gdansk, Poland) containing 0.5 µg/mL of ethidium bromide (Merck Life Science, Poznan, Poland). PCR products were visualized and photographed using a gel image system (GelDoc-It2 Imager, Analityk Jena US LLC, Upland, CA, USA).

Kwiatkowski P., Sienkiewicz M., Pruss A., Łopusiewicz Ł., Arszyńska N., Wojciechowska-Koszko I., Kilanowicz A., Kot B, & Dołęgowska B. (2022). Antibacterial and Anti-Biofilm Activities of Essential Oil Compounds against New Delhi Metallo-β-Lactamase-1-Producing Uropathogenic Klebsiella pneumoniae Strains. Antibiotics, 11(2), 147.

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Screening for Biosurfactant and Polysaccharide Genes

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Screening for the presence of surfactin, lichenysin, and levan genes was carried out using the primers listed in Table S1 (Supplementary material). Primer sequences were found in the NCBI database using Primer-BLAST. The PCR reaction was performed in a final volume of 12 µl containing 6.25 µl StartWarm HS-PCR Mix (A&A Biotechnology, Gdynia, Poland), 1.0 µl forward primer, 1.0 µl reverse primer, 2.75 nuclease free sterile H₂O (A&A Biotechnology) and 1 µl DNA of each strain. Thermal cycling was carried out in the Agilent Technologies SureCycler 8800 (Agilent Technologies, USA) with an initial denaturation of 95 °C for 5 min followed by 30 cycles at 95 °C for 1 min, 56–64 °C at 1 min, and 72 °C for 1 min, followed by a final extension of 72 °C for 5 min. Each amplification product was analyzed by electrophoresis using a 1% agarose gel followed by MIDORI Green Advance (Nippon Genetics Europe) staining and UV visualization. GeneRuler 1 kb DNA Ladder was used as a marker (ThermoFisher Scientific).

Shaimerdenova U., Kaiyrmanova G., Lewandowska W., Bartoszewicz M., Swiecicka I, & Yernazarova A. (2024). Biosurfactant and biopolymer producing microorganisms from West Kazakhstan oilfield. Scientific Reports, 14, 2294.

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Validation of Differentially Expressed Genes

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The characterization of SE events in the genes chosen for the validation of DASs was conducted with the use of Labcycler 48s (Syngen Biotech, Poland) and StartWarm HS-PCR Mix (A&A Biotechnology, Poland). The reaction mixture, in a final volume of 25 µL, contained 12.5 µL of Hot Start PCR Mix, primers (forward and reverse), nuclease-free deionized water, and 30 ng of cDNA. In non-template control, cDNA was substituted by water, or the reverse transcription step was omitted. PCR conditions and the primer sequences of chosen DASs are detailed in Table 1. The obtained PCR products were analysed on 1.5% agarose gels containing Midori Green Advance dye (Nippon Genetics Europe, Germany).

Dobrzyn K., Kiezun M., Kopij G., Zarzecka B., Gudelska M., Kisielewska K., Zaobidna E., Makowczenko K.G., Dall’Aglio C., Kamiński T, & Smolińska N. (2024). Apelin-13 modulates the endometrial transcriptome of the domestic pig during implantation. BMC Genomics, 25, 501.

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16S rRNA Amplicon Profiling for Bacterial DNA

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DNA isolates were a matrix for molecular biology techniques, such as end-point PCR for 16S rRNA fragments of bacterium nucleic acid (size ca. 550 bp). Amplification was performed with Start-Warm HS-PCR Mix (A&A Biotechnology, Gdynia, Poland), ddWater (aseptic, free from nucleases water, treated with DEPC), and appropriate primer sequences (F: sens 5' -TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG -3', R: antisens 5'-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAC ACA GGA CTA CHV GGG TAT CTA ATC C-3'.) selected from Kindworth et al. [22] , as the most promising bacterial primer pair for NGS. PCR protocol (95 °C for 3 min, 25 x (95 °C for 30s, 55 °C for 30s, 72 °C for 30s), 72 °C for 5 min, 4 °C holding) was performed on Sensoquest LabCycler (SensoQuest, Gemrany). Separation of PCR products on 2% agarose gel (Sigma-Aldrich, Germany) stained with Midori Green (Nippon Genetics Europe GmbH, Germany) by electrophoresis at 90V for 45 minutes. The results of the PCR amplicons were visualized under UV light in Gel Logic System 100 (Kodak Imaging System, Inc., USA). Amplification products (550 bp size) with a strong signal, and the high quality was chosen for further metagenomic investigation by Illumina MySeq (22 samples).

Dunaj J., Drewnowska J., Moniuszko-Malinowska A., Swiecicka I, & Pancewicz S. (2021). First metagenomic report of Borrelia americana and Borrelia carolinensis in Poland - a preliminary study. Annals of agricultural and environmental medicine : AAEM, 28(1).

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Screening Bacillus virulence genes by PCR

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Genomic DNA was extracted from an overnight culture of an isolate in Luria-Bertani (LB) broth using a DNeasy blood and tissue kit (Qiagen GmbH, Hilden, Germany) in accordance with the manufacturer’s protocol for Gram-positive bacteria. To amplify the nheA and hblA genes as well as cytK-1 and cytK-2 forms, the primer pairs listed in Table S3 were used as recommended in other works (50 (link)– (link)52 (link)). Due to the lack of full specificity of CKF/CKR for many cytK-2 target genes, the cytK sequences from the NCBI data bank (https://www.ncbi.nlm.nih.gov/) were used to design an alternative pair of primers (CytK2F/CytK2R). All B. cereus sensu lato isolates (n = 1,012) were screened for the presence of three virulence genes. The final PCR mixture (15 μl) contained 7.5 μl of StartWarm HS-PCR mix (A&A Biotechnology, Gdynia, Poland), 100 ng of DNA, and 0.25 μM each primer from a particular pair. B. cytotoxicus NVH 391/98 and B. cereus ATCC 14579 were applied as reference strains. PCR products were separated on a 1% agarose gel with Midori Green Advance DNA stain (Nippon Genetics Europe GmbH, Düren, Germany) and a GeneRuler 1 kb DNA ladder (Thermo Fisher Scientific) and visualized using a molecular gel imager, ChemiDoc XRS+ (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Drewnowska J.M., Stefanska N., Czerniecka M., Zambrowski G, & Swiecicka I. (2020). Potential Enterotoxicity of Phylogenetically Diverse Bacillus cereus Sensu Lato Soil Isolates from Different Geographical Locations. Applied and Environmental Microbiology, 86(11), e03032-19.

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Validation of Expression Profiling Genes

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We performed validation of the DASs for the 6 chosen genes—F-Box protein 15 (FBXO15); phosphatidylinositol glycan anchor biosynthesis class N (PIGN); peptidylglycine alpha-amidating monooxygenase (PAM); cleavage- and polyadenylation-specific factor 7 (CPSF7); cell division cycle 45 (CDC45); and reversion-inducing cysteine-rich protein with Kazal motifs (RECK)—using Labcycler 48 s (Syngen Biotech, Wroclaw, Poland). The PCR reaction was carried out using the StartWarm HS-PCR Mix (A&A Biotechnology, Gdansk, Poland). The reaction mixtures, at a final volume of 25 µL, consisted of 12.5 µL of Hot Start PCR Mix, 400 nM of forward and reverse primers, 15 ng of cDNA, and nuclease-free water. The primer sequences and the conditions of the PCR reactions are described in Supplementary Table S6. The final amplicons were detected using 1.5% agarose gels with the addition of Midori Green Advance (Nippon Genetics Europe, Düren, Germany).

Kopij G., Kiezun M., Dobrzyn K., Zaobidna E., Zarzecka B., Rak A., Kaminski T., Kaminska B, & Smolinska N. (2024). Visfatin Affects the Transcriptome of Porcine Luteal Cells during Early Pregnancy. International Journal of Molecular Sciences, 25(4), 2339.

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Genetic Modification Analysis in Porcine Cells

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Prior to analysis of the introduced genetic modifications, DNA isolation from modified primary porcine kidney fibroblasts was performed using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD, USA). Then PCR reactions were performed using the StartWarm HS-PCR Mix (A&A Biotechnology, Gdynia, Poland) to amplify DNA fragments that include modified loci and DNA fragments containing potential off-target sites. The purified PCR products were sequenced in the Sequencing Laboratory of the Faculty of Biology at the Adam Mickiewicz University in Poznan.

Ryczek N., Hryhorowicz M., Lipiński D., Zeyland J, & Słomski R. (2020). Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation. Genes, 11(6), 713.

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