Genomic DNA was used as a template to amplify sequences of the 16S rRNA genes of all species, rpoA and dnaA genes of Leuconostoc spp., and pheS and tuf genes of Lactobacillus spp. using the primers listed in Table 1 . Reactions were carried out in 50 μl, containing 10 pmol of each primer, 200 µM of each deoxynucleoside triphosphate (Thermo Scientific), 10 µl of 5× One Taq Standard Reaction buffer, 1.25 U One Taq Hot Start DNA polymerase (Thermo Scientific), and 100 ng template genomic DNA. PCR reactions were performed in a programmable thermal cycler (MultiGene OptiMax, Labnet International, Whitehead Scientific) with an initial denaturation step (94°C, 30 s), followed by 30 cycles of denaturation (94°C, 30 s), primer annealing, and elongation (see Table 1 ). Cycling was completed by a final elongation step (68°C, 10 min), followed by cooling to 4°C. The amplified fragments were purified using the DNA Clean and Concentrator™‐25 kit (Zymo Research, Inqaba Biotechnical Industries) according to the manufacturer's instructions.
Deoxynucleoside triphosphate
Manufactured by Thermo Fisher Scientific
Sourced in Lithuania, United States, Canada
Deoxynucleoside triphosphates (dNTPs) are essential building blocks for DNA synthesis. They are used as substrates by DNA polymerase enzymes to replicate DNA during cellular processes such as DNA replication and DNA repair.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase, by Thermo Fisher Scientific (3 mentions)
Control sirna, by Santa Cruz Biotechnology (2 mentions)
Revertaid m mulv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Oligo dt, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Dmem medium, by Merck Group (2 mentions)
Fumonisin b1 fb1, by Merck Group (2 mentions)
Imipramine, by Merck Group (2 mentions)
Onetaq hot start dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dna clean and concentrator 25 kit, by Zymo Research (1 mentions)
Multigene optimax, by Labnet (1 mentions)
Gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
Alicator lic cloning and expression kit 4, by Thermo Fisher Scientific (1 mentions)
Optitaq dna polymerase, by EURx (1 mentions)
Phusion hf buffer, by Thermo Fisher Scientific (1 mentions)
Nuclease free water, by Thermo Fisher Scientific (1 mentions)
Phusion hf dna polymerase, by Thermo Fisher Scientific (1 mentions)
15 protocols using deoxynucleoside triphosphate
1
Amplification and Purification of Bacterial Genes
2
Cloning and Verification of PCR Products
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Products of the PCR conforming to the expected size were purified from agarose gel using a Gel and PCR Clean-up Kit (Macherey-Nagel) and cloned into a pLATE52 vector which was part of the aLICATOR LIC Cloning and Expression Kit 4 (Thermo Scientific) according to the manufacturer’s instructions. Ligation products were used to transform E. coli NZY5a (NZYTech, Lisbon, Portugal). The presence of inserts of the expected size was confirmed by a colony PCR with LICfor/LICrev primers and sequencing. Briefly, an individual colony was picked up with a sterile pipette tip and transferred to a replica culture plate to save the clone for repropagation. The remaining cells were resuspended in 20 μL of reaction mixture containing 0.3 mM of each of the LICfor and LICrev primers (Table 1 ), 0.2 mM of each deoxynucleoside triphosphate (Thermo Scientific), 1× Pol C buffer and 0.5 U of OptiTaq DNA Polymerase (EURx Ltd., Gdansk, Poland). The thermal profile of the reaction was as follows: 95°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1.5 min, and a final elongation step at 72°C for 10 min. After the PCR was complete, 5 μL of each reaction was subjected to electrophoresis on a 1.5% agarose gel (data not shown).
3
Amplification of BoHV-6 DNA Fragments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To amplify BoHV-6 DNA fragments, 0.5 μg of DNA was used as a template in separate reactions containing 0.5 mM of each the primers specific for the gB (gBF/gBR) and gH (gHF/gHR) genes (Table 1 ), 0.2 mM of deoxynucleoside triphosphate (Thermo Scientific, Vilnius, Lithuania), 4 μL of 5× Phusion HF buffer, 0.4 U of Phusion HF DNA Polymerase (Thermo Scientific) and nuclease free water (Thermo Scientific) to a final volume of 20 μL. The thermal profile of the reaction was as follows: 98°C for 30 s; 35 cycles of 98°C for 10 s, 60°C for 20 s and 72°C for 1.5 min, and a final elongation step at 72°C for 10 min. The products of the first reaction were reamplified using the same conditions with pL52gBF/pLgBR and pL52gHF/pL52gHR primers (Table 1 ).
4
PCR detection of Hcp100 gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two sets of specific primers targeting a fragment of the Hcp100 gene were used (Bialek et al., 2002 (link); Porta et al., 1999 (link)). The conditions of the assay for organic fertilizers were described by Gómez et al. (2018) (link) (Gómez et al., 2018 (link)). In the first reaction, 10μl of DNA was added to 50μl total reaction mix with a final concentration of 10 mM Tris - HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 1U Taq polymerase (Thermo Scientific, Ref: EP0402. Waltham, M A, USA), 0.2 mM of each primer (HcI-HcII), and 0.2mm deoxynucleoside triphosphate (Thermo Scientific, Ref: R0181. Waltham, M A, USA). The mixture for the nested PCR was similar, except using 2μl of the product of the first PCR and 0.2mM of the inner primers (HcIII-HcIV). Temperatures and times for the first reaction, containing external primers HcI-HcII were one cycle at 94 °C for 5 min; 35 cycles at 94 °C for 30s, 66 °C for 1 min, and 72 °C for 1 min, and then a final cycle of 72 °C for 5 min. For the second step, the reaction consisted of a cycle at 94 °C for 5 min; 35 cycles at 94 °C for 30s, 65 °C for 30s, and 72 °C for 1 min, and then a final extension cycle at 72 °C for 5 min. Synthesis of the primers was performed by Integrated DNA Technologies (IDT, Coralville, IA, USA) (Gómez et al., 2018 (link)).
5
cDNA Amplification via PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One microliter of cDNA (100 ng of total RNA) was used in PCR reaction with a final concentration of 1 mmol/L magnesium chloride, 0.5 mmol/L deoxynucleoside triphosphate (Fermentas, Vilnius, Lithuania), 0.2 units of Taq DNA polymerase (Fermentas, Vilnius, Lithuania), and 0/3 μl of each cloning primer set 1 in a final volume of 20 μl. The PCR condition was 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 45 s.
Cycling was terminated with a final extension step of 7 min at 72°C. PCR products were visualized on a 1% agarose/green viewer gel.
Cycling was terminated with a final extension step of 7 min at 72°C. PCR products were visualized on a 1% agarose/green viewer gel.
6
DNA Extraction and Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Restriction endonuclease enzymes and silica-based DNA gel extraction kits were purchased from Fermentas (Thermo Fisher Scientific, Inc., Waltham, MA). PCR purification kit and DNA extraction kit were purchased from Bioneer (Bioneer, Korea). Deoxynucleoside triphosphate was purchased from Fermentas (Fermentas, Germany). The 10 × thermophilic buffer, pfu DNA polymerase, and MgSO4 got from Promega (Promega, Germany). Diaminobenzidine and nitrocellulose membrane were purchased from Sigma (Sigma, Germany) and Schleicher (USA). Oligonucleotides were used as primers for PCRs synthesis by Bioneer and ShineGene Molecular Biotech, Inc. (China).
7
Simultaneous Detection of LAA Pathogenicity Modules
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Simultaneous detection of the modules I, II and III of the LAA pathogenicity island was performed through PCR assay (Fig. S2 ). The primers LAA1_for +LAA1_rev and LAA2_for +LAA2_rev were designed to amplify modules I and II, respectively. A third pair of primers (ms3_for +ms3_rev), reported by Girardeau et al.35 (link), were included for the amplification of module III. The amplification reactions were performed in a final volume of 25 µL containing template DNA, 0.3 µM each primer, 0.4 µM each deoxynucleoside triphosphate (Fermentas, Lithuania), 5 µL 5X GoTaq DNA polymerase buffer and 1.25 U GoTaq DNA polymerase (Promega, USA). The amplification reaction included initial denaturing at 94 °C for 5 min, 30 cycles of denaturing at 94 °C for 30 s, hybridizing at 62.5 °C for 40 s and extension at 68 °C for 2 min, with a final extension at 72 °C for 10 min. PCR products were analyzed by electrophoresis in 1% agarose gel using Tris-acetate-EDTA buffer and stained with ethidium bromide.
8
Optimizing Hemolysin Gene Detection by PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The optimization of PMA treatment was performed via end-point PCR using oligonucleotides targeting a hemolysin gene (Wang et al., 1997) . Polymerase chain reaction was performed in a solution containing 1.5 U of Taq DNA polymerase (Fermentas, St. Leon-Rot, Germany), 1× PCR buffer (Fermentas), 3 mM MgCl 2 , 0.2 mM of each deoxynucleoside triphosphate (Fermentas), 0.8 μM of each primer (Invitrogen, São Paulo, Brazil) , and 1 μL of genomic DNA, in a final volume of 25 μL. The reactions were run on a Thermocycler (MiniCycler MJ Research, Watertown, MA) at 95°C for 3 min followed by 35 cycles at 95°C for 10 s, 56°C for 15 s, 72°C for 15 s, and 72°C for 3 min. The PCR products were visualized on 1% agarose gel (Invitrogen) in Trisborate-EDTA buffer with ethidium bromide (0.5 μg/ mL; Ludwig Biotecnologia Ltda, Porto Alegre, Brazil) under UV light.
The band intensity was determined using Quantity One 4.6.3 Software (BioRad Laboratories, Hercules, CA). The differences of band intensities between groups were analyzed by Student's t-test using IBM SPSS Statistics version 20 (Somers, NY). A P-value < 0.05 was considered statistically significant.
The band intensity was determined using Quantity One 4.6.3 Software (BioRad Laboratories, Hercules, CA). The differences of band intensities between groups were analyzed by Student's t-test using IBM SPSS Statistics version 20 (Somers, NY). A P-value < 0.05 was considered statistically significant.
9
Synthesis and Characterization of Gold Nanoparticles
10
Detailed ZMYND Fragment Amplification and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Selected ZMYND fragments were PCR-amplified from the genomic DNA or cDNA. PCR primers, designed with the use of Primer3 software, flanked whole exons or those exon fragments where the genotyped mutations were localized; to check if there were no SNPs in the primer binding sites, an SNPcheck v3 was used. The amplicon size for HRM analysis and sequencing, respectively, was 90–150 bp or 250–300 bp. A standard PCR amplification preceding dideoxy sequencing or cDNA analysis was carried out using: 0.4 ng/μl of DNA or cDNA template, 0.4 pM of each of the primers (forward and reverse), 0.12 mM deoxynucleoside triphosphates (Invitrogen) and a GoTaq polymerase kit (Promega); PCR reactions were conducted for 32 cycles with annealing temperatures of 60°C or 61°C. Real-time PCR reactions used in HRM analysis are described in one of the following sections. All primer pairs are listed in S1 Table .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!