Phire hot start 2

Manufactured by Thermo Fisher Scientific

The Phire Hot Start II is a high-performance DNA polymerase designed for efficient and accurate PCR amplification. It features a hot-start mechanism that prevents non-specific amplification, allowing for increased specificity and sensitivity in PCR reactions.

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Lab products found in correlation

Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions) Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Rq1 rnase free dnase, by Promega (1 mentions) Lysostaphin, by Merck Group (1 mentions) Phusion dna polymerase, by New England Biolabs (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) T4 ligase, by New England Biolabs (1 mentions)

Close spelling variants of this product

Phire Hot Start II DNA Polymerase Phire Hot Start II polymerase Phire Green Hot Start II DNA Polymerase Phire Hot Start II DNA Polymerase Kit Phire Green Hot Start II PCR Master Mix PCR Phire Hot Start II DNA polymerase Plant Phire Hot Start II DNA Polymerase Phire Hot Start II PCR Master Mix Phire Green Hot Start II Mastermix Phire II

4 protocols using phire hot start 2

Total RNA Extraction and cDNA Synthesis

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Total RNA was extracted from cells using the TRIzol reagent and treated with RQ1 RNase-free DNase (Promega) to remove potential genomic DNA contamination. cDNA synthesis was carried out using the Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) and random primers. For PCRs, either the Phire Hot Start II or Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) was used. Primers used in RT-PCR are listed in Supplemental Table 2.

Norppa A.J., Kauppala T.M., Heikkinen H.A., Verma B., Iwaï H, & Frilander M.J. (2018). Mutations in the U11/U12-65K protein associated with isolated growth hormone deficiency lead to structural destabilization and impaired binding of U12 snRNA. RNA, 24(3), 396-409.

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Staphylococcus aureus Genetic Manipulation

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Bacterial strains/plasmids and primers (IDT) used are described in Supplementary Table 2 and Supplementary Table 3, respectively. S. aureus were routinely grown on BHI agar (Difco), TSB (Oxiod) or Luria Broth (LB) at 37 °C with shaking at 200 rpm. For the selection of pIMAY-Z or pIMC8-YFP containing strains, BHI agar was supplemented with 10 μg ml⁻¹ chloramphenicol and 100 μg ml⁻¹ X-gal (BHIA-CX). For the protein expression, Terrific Broth (TB) was used (10 g l⁻¹ tryptone, 24 g l⁻¹ yeast extract, 10 g l⁻¹ glucose, 0.17 M KH₂PO₄ and 0.72 M K₂HPO₄). Restriction enzymes, Phusion DNA polymerase and T4 DNA ligase were purchased from New England Biolabs. Phire Hotstart II (for colony PCR) was purchased from Thermo Fisher. Genomic DNA from S. aureus was isolated from 1 ml of an overnight culture (DNeasy Blood and Tissue Kit—Qiagen) pretreated with 100 μg of lysostaphin (Sigma cat. no. L7386). lysostaphin sensitivity assays were performed as described³².

Monk I.R., Shaikh N., Begg S.L., Gajdiss M., Sharkey L.K., Lee J.Y., Pidot S.J., Seemann T., Kuiper M., Winnen B., Hvorup R., Collins B.M., Bierbaum G., Udagedara S.R., Morey J.R., Pulyani N., Howden B.P., Maher M.J., McDevitt C.A., King G.F, & Stinear T.P. (2019). Zinc-binding to the cytoplasmic PAS domain regulates the essential WalK histidine kinase of Staphylococcus aureus. Nature Communications, 10, 3067.

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Recombinant Expression of Isopentenyl Monophosphate Kinase

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Isopentenyl monophosphate kinase (IPK) from Thermoplasma acidophilum DSM 1728^{[6 (link), 9 (link)]} was codon-optimized and synthesized by Genewiz, Inc (see Supplemental Table S1). The ipk gene was PCR amplified from the synthesized template and sub-cloned into pET28a using NdeI and XhoI restriction sites to generate an N-terminally hexa-histidine tagged fusion. Briefly, PCR was performed using Phire Hot Start II polymerase (ThermoFisher) according to the supplier’s protocol, purified by agarose gel electrophoresis, digested with NdeI and XhoI, and purified again. The digested PCR product and similarly treated pET28a were ligated at room temperature with T4 ligase (New England BioLabs) according to the supplier’s protocol. The ligation mixture was then transformed into E. coli DH5α and plated onto LB agar plates containing 50 μg/mL kanamycin. Individual colonies were picked, grown in the presence of kanamycin, plasmids purified and the ipk gene sequence verified by DNA sequencing (Genewiz).

Lund S., Courtney T, & Williams G.J. (2019). Probing the substrate promiscuity of isopentenyl phosphate kinase as a platform for hemiterpene analogue production. Chembiochem : a European journal of chemical biology, 20(17), 2217-2221.

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In Vitro Transcription of RNAs

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All RNAs used in this study were prepared by in vitro transcription (IVT) using standard protocol. As an IVT template PCR products were used obtained in the reaction with Phire Hotstart II polymerase (Thermo Fisher) according to manufacturer’s instructions (RyhB), or hybridized DNA oligonucleotides (CyaR, 3ʹETS^leuZ; Sigma). The sequences of used primers and DNA oligonucleotides are summarized in Table S1. RNA was separated on 6% polyacrylamide denaturing gel (National Diagnostics), electroeluted in TBE (Whatman Elutrap) and cleaned up using PureLinkTM RNA Microscale Kit (Invitrogen).

Dendooven T., Sinha D., Roeselová A., Cameron T.A., De Lay N.R., Luisi B.F, & Bandyra K.J. (2021). A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation. Molecular Cell, 81(14), 2901-2913.e5.

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