Green gotaq reaction buffer

Manufactured by Promega

Sourced in United States

Green GoTaq Reaction Buffer is a ready-to-use buffer solution designed for use with the GoTaq DNA Polymerase in PCR (Polymerase Chain Reaction) applications. The buffer contains dNTPs, MgCl2, and a green dye for easy visualization of the reaction mixture.

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Lab products found in correlation

Gotaq dna polymerase, by Promega (16 mentions) Gotaq g2 dna polymerase, by Promega (8 mentions) Gotaq green master mix, by Promega (4 mentions) Mgcl2, by Promega (4 mentions) Dntps, by Promega (3 mentions) Pcr nucleotide mix, by Promega (3 mentions) Exo sap treated, by Thermo Fisher Scientific (2 mentions) Dna ladder, by New England Biolabs (2 mentions) Taq polymerase, by Promega (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Gotaq g2 flexi dna polymerase, by Promega (2 mentions) Qubit fluorometer, by Thermo Fisher Scientific (2 mentions) Qiaquick gel extraction kit, by Qiagen (2 mentions) Dntp mix, by Thermo Fisher Scientific (2 mentions) Pureyield plasmid miniprep system, by Promega (2 mentions) Colorless gotaq reaction buffer, by Promega (2 mentions) Powersoil dna isolation kit, by Qiagen (2 mentions) Dneasy plant mini kit, by Qiagen (2 mentions) Dna polymerase, by Promega (1 mentions) Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)

46 protocols using green gotaq reaction buffer

Genomic DNA Extraction and PCR Analysis of CAG Repeats

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Genomic DNA was isolated from Can^R clones using a previously described method⁶⁴. Cells were resuspended in 1.5 µL of 0.5 mg/mL lyticase solution [0.9 M Sorbitol, 0.1 M EDTA (pH 7.4)] in microplates, incubated at 37°C for 15 min, then resuspended in 50 µL of water. The samples were incubated at 100°C for 5 min and centrifuged at 2,500 × g for at least 2 min. For PCR analysis of CAG repeat length, reactions included 1× Green GoTaq reaction buffer (M7911, Promega), 0.16 mM dNTP mix, 0.8 µM of each primer, 0.5 units of Taq DNA polymerase (SibEnzyme or Thermo Scientific), and 1 µL of the DNA supernatant in a 12.5 µL total reaction volume. Primers JK316 and JK317 result in a 544 bp product for (CAG)₁₄₀. Primers JK318 and JK319 result in a 625 bp product for (CAG)₁₄₀ (Figure 1B). Primers JK153 and JK171 result in a 462 bp product for (CAG)₁₄₀ and 321 bp (CAG)₉₃. 10 µL PCR products were run on 1.5% agarose in 0.5× TBE alongside 50 bp and 100 bp DNA ladders (NEB). PCR products were sized using TotalLab Quant software for 1D DNA gels.
The same PCR method was used to generate products for sequencing the CAG repeats (FlankL and CanF) or the CAN1 gene (JK167 and JK168, JK169 and JK170). PCR products were Exo-SAP treated (Affymetrix) and sequenced by Eton Bioscience or University of Chicago Sequencing Core).

Kim J.C., Harris S.T., Dinter T., Shah K.A, & Mirkin S.M. (2016). The role of break-induced replication in large-scale expansions of (CAG)n•(CTG)n repeats. Nature structural & molecular biology, 24(1), 55-60.

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PCR Amplification of Late RT Products

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Late RT PCR products were amplified by PCR using the pR 5′-AGACCAGATCTGAGCCTGGGAG-3′ and pMA’ 5′-CTGACGCTCTCGCACCC-3′ primers at a final concentration of 100 nM for each primer [21 (link)]. 400 ng DNA, extracted from LAI-infected or uninfected control cells, was used as template in each PCR reaction containing 1× Green GoTaq Reaction buffer (Promega), 200 µM PCR nucleotide mix (Promega), 100 nM pR primer, 100 nM pMA’ primer, and Go Taq G2 DNA polymerase (M7841 Promega) at a final concentration of 1.25 U, in a reaction volume of 50 µl. The PCR cycling conditions were as follows; 95 °C, 2 min; 40 cycles at 95 °C, 30 s; 64 °C, 30 s; 73 °C, 30 s; followed by 73 °C for 5 min.

Ingemarsdotter C.K., Zeng J., Long Z., Lever A.M, & Kenyon J.C. (2018). An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation. Retrovirology, 15, 25.

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Miscanthus Leaf RNA Extraction and PCR

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Developing Miscanthus leaves were cut at their node, dissected, and immediately frozen in liquid nitrogen. Total RNA was isolated from ground leaf samples using a combination of Trizol Reagent (Invitrogen) and the Qiagen RNAeasy Plant Mini Kit. RNA quantity was assessed using an Epoch Microplate Spectrophotometer (BioTEK). The quality and integrity of total RNA were evaluated on an agarose gel. cDNA was prepared using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen) with oligo(dT) primers. PCR was performed using the Green GoTaq Reaction Buffer and DNA polymerase (Promega) with the following thermal cycling steps: 95 °C for 2 min, 35 cycles of 30 s at 95 °C, 1 min at 60 °C, and 30 s at 72 °C, final extension 72 °C for 5 min. RT-PCR products were analysed on agarose gels with Zea mays spermine synthase1 (ZmSPMS1) as a reference gene. The primers used for PCR amplification of genomic and complementary DNA from transgenic and wild-type control plants are listed in Additional file 1: Table S2.

Bhatia R., Timms-Taravella E., Roberts L.A., Moron-Garcia O.M., Hauck B., Dalton S., Gallagher J.A., Wagner M., Clifton-Brown J, & Bosch M. (2023). Transgenic ZmMYB167 Miscanthus sinensis with increased lignin to boost bioenergy generation for the bioeconomy. Biotechnology for Biofuels and Bioproducts, 16, 29.

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Sanger Sequencing PCR Amplification

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To amplify the fragments of interest for Sanger sequencing, 25μl PCR reactions containing template genomic DNA, 20 μmoles of each of the forward and reverse primers, 2.5 μM dNTPs (Promega, Madison, Wisconsin, USA), 1.5 nm MgCl2, 1X Green GoTaq® Reaction Buffer (Promega, Madison, Wisconsin, USA) and 0.01U GoTaq® G2 Flexi DNA Polymerase (Promega, Madison, Wisconsin, USA) was used. Amplification was performed in an ABI 2720 Thermal Cycler (Applied Biosystems Inc., Foster City, California, USA). To visualize the PCR amplicons and to investigate if non-specific primer binding or contamination was present, agarose gel electrophoresis was used. PCR amplicons were visualized using a SynGene UV gel documentation system (Synoptics Ltd., Cambridge, UK) with GeneTools software version 3.0.6 (Synoptics Ltd., Cambridge, UK).

Sebate B., Cuttler K., Cloete R., Britz M., Christoffels A., Williams M., Carr J, & Bardien S. (2021). Prioritization of candidate genes for a South African family with Parkinson’s disease using in-silico tools. PLoS ONE, 16(3), e0249324.

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Gene-Specific Primer Design and PCR Optimization

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Gene-specific primers were designed using the program PrimerQuest from IDT (http://eu.idtdna.com/PrimerQuest/Home/Index) to amplify between 150–1200 bp of genomic sequence (S1 and S2 Tables). Unless stated otherwise, PCR conditions were as follows: PCR was carried out in individual 0.2 mL polypropylene PCR tubes (Axygen^®, Corning^®, USA) containing 3 μL cDNA template, 10 μM of each primer, 4 μL of 5 × Green GoTaq^® reaction buffer (Promega, USA), 0.2 μL GoTaq^® DNA Polymerase (5 U/μL, Promega, USA), 1 μL dNTP (5 mM of each dNTP, Invitrogen, Life Technologies, USA) in a final volume of 20 μL. The PCR programme consisted of an initial 2 min step at 94°C, followed by 25 cycles of: 1) 15 sec at 94°C; 2) 15 sec at 50°C; and 3) 1 min and 30 sec at 72°C, followed by a final extension step of 10 min at 72°C. Negative controls containing RNA template and a positive control containing genomic DNA were subjected to the same procedure to exclude any possible contamination or to detect PCR inhibitors. To analyse PCR amplified fragments, 15 μL of the final PCR product was run on a 1.5% agarose (UltraPure^™, Invitrogen, Life Technologies, USA), 1 x TBE (50mMTris-Cl pH 8, 50mM Boric Acid, 1mM EDTA) gel.

Calixto C.P., Simpson C.G., Waugh R, & Brown J.W. (2016). Alternative Splicing of Barley Clock Genes in Response to Low Temperature. PLoS ONE, 11(12), e0168028.

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Insect DNA Isolation and cCENH3 Amplification

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Total DNA was isolated from ~50 mg of adult insects (25 individuals) by using DNeasy Blood and Tissue Kit (Qiagen). DNA quantification was done by Qubit fluorometer (Invitrogen) using Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific). Based on the cCENH3 coding sequence and its flanking regions, the primers cCENH3pr0 (5'- AAGGTAGGATAATGCCGA-3'), cCENH3pr1 (5'-ATGGCCCGTTCTAAG-3'), cCENH3pr3 (5'-TTAACCACCTTCTTTTTCC-3’), and cCENH3pr5 (5'-TTAACCACCTTCTTTTTCCCTCC-3’) were designed to amplify the entire cCENH3 gene sequence. PCR reaction was performed in 50-μl volume containing 1× Green GoTaq Reaction Buffer (Promega), 2.5 mM MgCl₂, 0.1 mM of each dNTP, 0.1 μM of each primer and 2 units of GoTaq G2 DNA Polymerase (Promega). PCR program included predenaturation at 94°C for 3 min, 35 cycles od amplification (denaturation at 94°C for 20 s, annealing at 61°C for 20 s, and extension at 72°C for 30 s), and final extension at 72°C for 7 min. PCR products were analyzed in agarose gel electrophoresis, extracted by QIAquick Gel Extraction Kit (Qiagen), and Sanger-sequenced in Macrogen Europe Laboratory (Amsterdam, The Netherlands).

Gržan T., Despot-Slade E., Meštrović N., Plohl M, & Mravinac B. (2020). CenH3 distribution reveals extended centromeres in the model beetle Tribolium castaneum. PLoS Genetics, 16(10), e1009115.

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RNA Extraction and RT-PCR for Viral Subtype Detection

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Total RNA from the samples was extracted using the RNeasy^® Mini kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The M-MLV Reverse Transcriptase kit (Promega, Madison, WI, USA) was used to perform reverse transcription, following the manufacturer’s instructions. For the polymerase chain reaction (PCR), the G protein gene was used for the detection of subtypes A and B (Table 1), using the following reagents and concentrations: 2 mM magnesium chloride, 0.25 mM deoxyribonucleotide phosphates, 0.3 μM of each primer, 1 U of Taq DNA polymerase GoTaq^® DNA Polymerase (Promega, Madison, WI, USA), 1× Green GoTaq^® Reaction Buffer (Promega, Madison, WI, USA), 3 μL of sample and sterile ultrapure water, to make 25 μL. The reactions were carried out in a thermocycler, using the following cycling parameters: 94 °C for 2 min; 35 cycles of 94 °C for 30 s, 63 °C for 30 s, 68 °C for 3 min; and a final cycle of 72 °C for 10 min [21 (link)].
The samples were subjected to horizontal electrophoresis in 1% agarose gel, using GelRed (MilliporeSigma™, Burlington, MA, USA) as a DNA intercalating agent. Amplicon sizes were determined by comparison with the low-molecular-weight (LMW) marker.

Salles G.B., Pilati G.V., Savi B.P., Muniz E.C., Dahmer M., Vogt J.R., de Lima Neto A.J, & Fongaro G. (2023). Surveillance of Avian Metapneumovirus in Non-Vaccinated Chickens and Co-Infection with Avian Pathogenic Escherichia coli. Microorganisms, 12(1), 56.

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Genomic DNA Extraction and PCR Analysis of CAG Repeats

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16S rRNA Gene Amplification and Sequencing

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Genomic DNA was extracted from 100 µL of liquid cultures incubated 10 min at 99°C in a thermal cycler and 10 min at −20°C for three times. These extractions were used to PCR-amplify the nearly complete 16S rRNA gene with primers 27Fmod (5′-AGR GTT TGA TCM TGG CTC AG-3′) and 1492Rmod (5′-TAC GGY TAC CTT GTT AYG ACT T-3′) from Page et al. (76 ). Each PCR reaction was composed of 32.75 µL Milli-Q water, 10 µL 5× Green GoTaq Reaction Buffer (Promega), 1 µL dNTPs mix (each deoxynucleotide at 10 mM), 2 µL of each primer (10 µM), 0.25 µL GoTaq DNA Polymerase (Promega), and 2 µL of extracted DNA. When faint or no bands were observed on agarose gel electrophoresis following PCR, DNA extraction was repeated with DNeasy Blood&Tissue Kit (Qiagen) following the manufacturer’s recommendations. Purification and OneShot Sanger sequencing of PCR products was carried out by Genoscreen (Lille, France) with the two above-mentioned primers.

Rey-Velasco X., Deulofeu-Capo O., Sanz-Sáez I., Cardelús C., Ferrera I., Gasol J.M, & Sánchez O. (2023). Expanding success in the isolation of abundant marine bacteria after reduction in grazing and viral pressure and increase in nutrient availability. Microbiology Spectrum, 11(5), e00890-23.

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DNMT3B Polymorphism Genotyping by PCR-RFLP

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Genomic DNA was isolated from buccal epithelial cells by standard procedure (Aidar and Line, 2007 (link)) and quantified by spectrophotometry. Genotypes were determined by PCR amplification followed by digestion with restriction endonuclease (PCR-RFLP) as described by Xiao et al. (2008) (link) with minor modifications. Conditions used to amplify a 380 bp fragment containing the polymorphism are shown in Table 1. Reactions were carried out in a final volume of 15 μL containing 50ηg genomic DNA, 1X Green GoTaq™ Reaction Buffer (with 1.5 mM MgCl₂), 9 pmol each primer, and 1 U GoTaq™ Polymerase (Promega). PCR products were digested with XmaJI endonuclease (Thermo Fischer Scientific) according to supplier recommendations, and the digested products were resolved in 2% agarose gel, ethidium bromide stained and visualized under UV light. Genotypes are identified according to the band pattern as shown in Table 1.Table 1

PCR-RFLP conditions and genotypes observed for DNMT3B −149C > T polymorphism.

Primers (5′-3′)*	PCR conditions	Band pattern (bp)
F: 5′-TGCTGTGACAGGCAGAGCAG-3′	94 °C for 5 min, followed by 32 cycles of 94 °C for 30 s, 60 °C for 50 s, and 72 °C for 1 min, and a final elongation step at 72 °C for 4 min	CC: 380
F: 5′-TGCTGTGACAGGCAGAGCAG-3′		CT: 380, 207 and 173
R: 5′-GGTAGCCGGGAACTCCACGG-3′		TT: 207 and 173

As described by Xiao et al. (2008) (link).

Moura C.M., Bastos P.R., Ribeiro J.S., Ribeiro M.G., Amorim M.R, & Costa-Lima M.A. (2017). DNA (cytosine-5)-methyltransferase 3B (DNMT 3B) polymorphism and risk of Down syndrome offspring. Saudi Journal of Biological Sciences, 25(1), 101-104.

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