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Hot start taq 2x master mix

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Hot Start Taq 2X Master Mix is a ready-to-use solution for PCR amplification. It contains Taq DNA polymerase, reaction buffer, magnesium chloride, and dNTPs.

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Lab products found in correlation

Dneasy powersoil extraction kit, by Qiagen (3 mentions) Miseq sequencing, by Illumina (3 mentions) Minelute pcr purification kit, by Qiagen (2 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Dreamtaq hot start pcr master mix kit, by Thermo Fisher Scientific (1 mentions) Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions) E gel, by Thermo Fisher Scientific (1 mentions) Gibson assembly kit, by New England Biolabs (1 mentions) Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions) Biorad s100 gradient thermal cycler, by Bio-Rad (1 mentions) Dneasy powersoil kit, by Qiagen (1 mentions) Miseq platform, by Illumina (1 mentions) Quick load 100 bp dna ladder, by New England Biolabs (1 mentions) T100 thermal cycler, by Bio-Rad (1 mentions) Zymobiomics microbial community standard, by Zymo Research (1 mentions) Q5 hot start high fidelity 2x master mix, by New England Biolabs (1 mentions) Geneamp pcr system 2700 thermal cycler, by Thermo Fisher Scientific (1 mentions) Iscript reverse transcription supermix, by Bio-Rad (1 mentions)

Close spelling variants of this product

One Taq Hot Start 2X Master Mix with Standard Buffer LongAmp Hot Start Taq 2X Master Mix Taq 2X Master Mix Hot Start Taq Taq Master Mix

17 protocols using hot start taq 2x master mix

1

Quantitative PCR Amplification and Library Preparation

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For PCR of individual
templates, 800 μL of amplification was performed using 1X Hot
Start Taq 2X Master Mix (New England Biolabs), 0.5 μM each forward
and reverse primer, and 2.5 nM template (see Table S5). The mixture was incubated at 95 °C for 5 min and
then subjected to 12 cycles of 95 °C for 40 s, 50 °C for
40 s, and 68 °C for 40 s.
For preparation of library pools
(L8, L14), a 20 μL extension reaction was first
performed using 1 μM each template primer and reverse primer,
1X KOD Plus buffer (Toyobo), 0.02 U/μL KOD plus DNA polymerase,
0.2 mM dNTPs, and 1.5 mM MgSO4. The mixture was incubated
at 95 °C for 2 min and then subjected to six cycles of 95 °C
for 40 s, 60 °C for 40 s, and 68 °C for 60 s. The entire
extension mix was then amplified in an 800 μL reaction containing
1X Hot Start Taq 2X Master Mix (New England Biolabs), 250 nM FW primer,
and 250 nM reverse primer.
All PCR products were confirmed by
gel electrophoresis (E-Gel,
ThermoFisher Scientific), purified using the MinElute PCR Purification
Kit (Qiagen), and quantified by UV absorbance (NanoDrop).
Chan A.I., Sawant M.S., Burdick D.J., Tom J., Song A, & Cunningham C.N. (2023). Evaluating Translational Efficiency of Noncanonical Amino Acids to Inform the Design of Druglike Peptide Libraries. ACS Chemical Biology, 18(1), 81-90.
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2

Microbial DNA Extraction and 16S Sequencing

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Microbial DNA was extracted from different body site samples using the Qiagen DNeasy PowerSoil extraction kits (Qiagen, Germantown, MD, USA) according to manufacturer’s protocol. Samples that did not have enough biomass were not able to be sequenced. The V4 variable region of the 16S rRNA gene was amplified with PCR primers 515f/806r [25 (link)] in a 30 cycle PCR using the DreamTaq Hot Start PCR Master Mix Kit (Thermoscientific, Waltham, MA, USA). PCR was performed in 20 μL vol and included 2 μL (7.5 μM concn) of forward and reverse primers, 12.5 μL of Hot Start Taq 2X Master Mix (New England BioLabs Inc., Ipswich, MA., USA), 3.5 μL of deionized water, and 2 μL of sample DNA. Thermal cycle conditions were 95 °C for 3 min for the initial denaturing step, followed by 30 cycles of 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min. PCR products were checked on a 2% agarose gel for correct product size formation (approx. 350 bp). The Michigan State University Genomics Core performed library preparation prior to Illumina MiSeq sequencing, following the manufacturer’s guidelines [25 (link)]. Reagent controls using certified DNA free water were run through library preparation and PCR and did not generate libraries. For quality control, samples submitted for sequencing included a random blank sample of technical replicates.
Beckers K.F., Gomes V.C., Crissman K.R., Liu C.C., Schulz C.J., Childers G.W, & Sones J.L. (2023). Metagenetic Analysis of the Pregnant Microbiome in Horses. Animals : an Open Access Journal from MDPI, 13(12), 1999.
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3

Quantitative Analysis of Angiogenic Factors

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Total RNA was isolated from cell culture using the PureLink RNA Mini Kit (Invitrogen, 12183018A). 500 ng of RNA from each sample was used to generate cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, K1621). qPCR was performed using Hot Start Taq 2x Master Mix (New England BioLabs, M0496L). Samples were probed using primer pairs for Beta Actin (Hs01060665_g1), VEGF (Hs00900055_m1), KDR/Flk-1 (Hs00911700_m1), Flt-1 (Hs01052961_m1), IDO1 (Hs00984148_m1), CD163 (Hs00174705_m1), and sFlt-1 (5’-3’ TGG GGA GGG GAG GAT GTT AG, 3’-5’ TAA GGG AGG TGC GTT GAA CC).
Engel J.E., Williams M.L., Williams E., Azar C., Taylor E.B., Bidwell G.L, & Chade A.R. (2020). Recovery of Renal Function Following Kidney Specific VEGF Therapy in Experimental Renovascular Disease. American journal of nephrology, 51(11), 891-902.
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4

DNA Transformation and Genetic Modifications

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Linear and plasmid DNA transformations were performed as described previously [23 (link)] as were transductions using the P1vir bacteriophage [24 (link)]. DNA constructs were created using the Gibson assembly kit (#E2611; New England Biolabs). Chromosomal gene deletions were generated using the λ Red recombinase system [25 (link)]. Unless otherwise stated, all PCR products were generated using E. coli MG1655 chromosomal DNA as a template and all strains were verified by PCR and/or sequence analysis (New England Biolabs DNA sequencing facility). PCR reactions used to generate sequencing templates or genetic constructions were performed with the Q5 High-Fidelity DNA Polymerase (#M0491; New England Biolabs) while diagnostic PCR reactions used the Hot Start Taq 2X Master Mix (#M0496; New England Biolabs). New England Biolabs produced all restriction enzymes and ligases used in these experiments.
Kingston A.W., Roussel-Rossin C., Dupont C, & Raleigh E.A. (2015). Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12. PLoS ONE, 10(7), e0130813.
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5

Bacterial Diversity Analysis from Sediments

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Bacterial DNA from sediment samples was extracted using DNeasy Power Soil Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA quantity was measured using NanoDrop Spectrophotometer (Thermo Fisher Scientific, USA), followed by dilution to 50 ng/μl final concentration. A total of 35 cycles of amplification for 50 µl final reactions were conducted in a BioRad S100 Gradient Thermal Cycler (Bio-Rad Laboratories, Inc., Foster City, California, USA) containing 2 µl template DNA, 1 µl of each V3-V4 sequencing primers (Part # 15,044,223 Rev. B), 25 µl of Hot Start Taq 2X Master Mix (New England BioLab Inc., USA) and 21 µl DEPC treated water. AMPure XP beads were used to process and clean amplified PCR products, and then, in accordance with the Illumina standard methodology (Part # 15044223 Rev. B), amplicon barcoding was performed using a secondary PCR. Samples were sequenced up to 50,000 reads on an Illumina MiSeq platform (Illumina Inc., USA) using a v3 kit (600 cycles, Part # MS-102-3003).
Nguyen T.T., Foysal M.J., Gupta S.K., Tay A., Fotedar R, & Gagnon M.M. (2024). Effects of carbon source addition in rearing water on sediment characteristics, growth and health of cultured marron (Cherax cainii). Scientific Reports, 14, 1349.
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6

Molecular Identification of Anopheles Mosquitoes

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Individual mosquitoes were identified to species level according to Santolamazza et al. (77 (link)). PCRs contained 2 μl of 10 μM forward primer (5′-TCGCCTTAGACCTTGCGTTA-3′), 2 μl of 10 μM reverse primer (5′-CGCTTCAAGAATTCGAGATAC-3′), 1 μl of extracted DNA, and 10 μl of Hot Start Taq 2X Master Mix (New England Biolabs, UK) for a final reaction volume of 20 μl. Prepared reactions were run on a Bio-Rad T100 thermal cycler with the following conditions: 10 min denaturation time at 94°C, followed by 35 amplification cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 60 s, followed by a final extension at 72°C for 10 min. PCR products were visualized on 2% E-gel agarose gels in an Invitrogen E-gel iBase real-time transilluminator. A Quick-Load 100-bp DNA ladder (New England Biolabs) was used to determine band size. Amplified PCR products of 479 or 249 bp were indicative of An. coluzzii or An. gambiae s.s., respectively. As the dominant species, only An. coluzzii individuals of the same age and resistance phenotype were selected and pooled for 16S rRNA sequencing.
Pelloquin B., Kristan M., Edi C., Meiwald A., Clark E., Jeffries C.L., Walker T., Dada N, & Messenger L.A. (2021). Overabundance of Asaia and Serratia Bacteria Is Associated with Deltamethrin Insecticide Susceptibility in Anopheles coluzzii from Agboville, Côte d’Ivoire. Microbiology Spectrum, 9(2), e00157-21.
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7

16S Rapid Sequencing Protocol for ISS

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Following removal from cold stowage, Hot Start Taq 2X Master Mix (NEB) containing 16S primers from the ONT 16S Rapid sequencing kit (SQK-RAS201) was pipetted into 10 ng of ZymoBIOMICS™ Microbial Community Standard (Zymo Research, Irvine, CA, USA) in three separate tubes of an 8 tube PCR strip. The reaction product was placed into a miniPCR (Amplyus, Cambridge, MA, USA) thermal cycler that was already onboard the ISS. PCR parameters were an initial denaturation of 95 °C for 1 min, followed by 30 cycles of 95 °C for 10 s, 60 °C for 10 s, 72 °C for 20 s, and a final extension of 72 °C for 90 s. All library preparations were completed in triplicate by pipetting with Eppendorf Research Plus pipettes on the open bench top. The amplified products were placed into the −80 to −90 °C MELFI dewar until they were used for library preparation.
Burton A.S., Stahl S.E., John K.K., Jain M., Juul S., Turner D.J., Harrington E.D., Stoddart D., Paten B., Akeson M, & Castro-Wallace S.L. (2020). Off Earth Identification of Bacterial Populations Using 16S rDNA Nanopore Sequencing. Genes, 11(1), 76.
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8

PCR and LAMP Reactions for ISS

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Complete reaction mixes were prepared for both the PCR and LAMP reactions slated for execution on the ISS. Each tube in an eight‐tube strip (Eppendorf 0030124359; Eppendorf) contained 25 μL of either Q5 Hot Start High‐Fidelity 2X Master Mix (NEB M0494; New England Biolabs, Inc) or Hot Start Taq 2X Master Mix (NEB M0496; New England Biolabs, Inc), along with 5 μL primers prepared at 5 μmol/L concentration, 18 μL distilled water, and 2 μL of 1 ng/μL pSPACETELO plasmid. Exact reaction conditions detailed in Table S1, primer sequences can be found in Table S2. Strips of tubes were stored at −125°C for 4 weeks and 8 weeks, thawed, and then immediately amplified. PCR amplification conditions for cold stowage stability study were as follows: 94°C/30 s [94°C/15 s, 50°C/15 s, 68°C/60 s] × 30 cycles, 68°C/2 min. LAMP amplification conditions for cold stowage stability study were as follows: 65°C/30 min. Results of cold stowage studies are shown in Table S1 and Figures S1 and S2.
Rubinfien J., Atabay K.D., Nichols N.M., Tanner N.A., Pezza J.A., Gray M.M., Wagner B.M., Poppin J.N., Aken J.T., Gleason E.J., Foley K.D., Copeland D.S., Kraves S, & Alvarez Saavedra E. (2020). Nucleic acid detection aboard the International Space Station by colorimetric loop‐mediated isothermal amplification (LAMP). FASEB bioAdvances, 2(3), 160-165.
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9

TMPRSS2-ERG Fusion Detection

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Total RNA was extracted from LNCaP and VCaP cells using TRIzoL (Thermo Fisher Scientific). RNA was reversely transcribed to cDNA via iScriptTM Reverse Transcription Supermix (Bio-Rad Laboratories). After quantification by NanoVue Plus spectrophotometer (GE Healthcare), 500 ng cDNA was utilized as a template for amplification of TMPRSS2-ERG fusion using Hot Start Taq 2X Master Mix (New England Biolabs) and GeneAmp PCR System 2700 thermal cycler (Applied Biosystems). The forward and reverse PCR primers for T1/E4 fusion included 5’-TAGGCGCGAGCTAAGCAGGAG-3’ and 5’-CCATAT TCTTTCACCGCCCACTCC-3’ (Integrated DNA Technologies). ERG isoform-6 primers were 5'-GGTACGAAAACACCCCTGTG-3' (forward) and 5'-CCAAATCAACAGAGGCAGAA-3' (reverse); the total ERG primers were 5'-AACGAGCGCAGAGTTATCGT-3' (forward) and 5'-GTGAGCCTCTGGAAGTCGTC-3' (reverse). The final volume was 25 μl, and an initial denaturation step of 95 °C for 5 min was followed by 40 cycles of 95 °C for 30 s, 59 °C for 30 s, 72 °C for 30 s, and one cycle at 72 °C for 5 min. T1/E4 fusion, isoform-6, and total ERG cDNA were detected by 2% agarose gel electrophoresis (supplemental Fig. S1).
Fu Z., Rais Y., Bismar T.A., Hyndman M.E., Le X.C, & Drabovich A.P. (2021). Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein by Orthogonal Immunoprecipitation–Mass Spectrometry Assays. Molecular & Cellular Proteomics : MCP, 20, 100075.
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10

Fecal Microbiome DNA Extraction and Amplification

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Microbial DNA was extracted from fecal samples using the Qiagen DNeasy PowerSoil extraction kits (Qiagen) according to manufacturer's protocol. The V4 variable region of the 16S rRNA gene was amplified with PCR primers 515/806 in a 30 cycle PCR using the DreamTaq Hot Start PCR Master Mix Kit (Thermoscieniftic). PCR was performed in 20 μl vol and included: 2 μl (7.5 μM concn) of forward and reverse primers, 12.5 μl of Hot Start Taq 2X Master Mix (New England BioLabs Inc.), 3.5 μl of deionized water, and 2 μl of sample DNA. Thermal cycle conditions were 95°C for 3 min for initial denaturing step, followed by 30 cycles of 95°C for 30 s, 50°C for 1 min, and 72°C for 1 min. PCR products were checked on a 2% agarose gel for correct product size formation (approx 350 bp). Michigan State University Genomics Core performed library preparation prior to Illumina MiSeq sequencing following the manufacturer's guidelines. Reagent controls using certified DNAfree water were run through library preparation and PCR and did not generate libraries. For quality control, samples submitted for sequencing included a random blank sample of technical replicates.
Beckers K.F., Schulz C.J., Flanagan J.P., Adams D.M., Gomes V.C., Liu C., Childers G.W, & Sones J.L. (2022). Sex‐specific effects of maternal weight loss on offspring cardiometabolic outcomes in the obese preeclamptic‐like mouse model, BPH/5. Physiological Reports, 10(17), e15444.
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