The pre-amplification products were directly used for a multiplex TSPE reaction. Primers were extended and biotin-14-dCTP (Thermo Fisher Scientific, Waltham, USA), instead of normal dCTP, was incorporated, alongside with the remaining unmodified nucleotides (dATP, dGTP, dTTP; Thermo Fisher Scientific). A set of eleven primers was used: two generic primers, to detect all tospoviruses (tTospo_GENs/as), eight specific primers, to identify four tospovirus species (tTSWVs/as, tINSVs/as, tWSMoVs/as and tCaCVs/as) and one plant internal control primer (tNad5) for nad5. TSPE mixes of 20 µl containing 5 µl of the pre-amplification products were prepared (0.75 U Platinum GenoTYPE Tsp DNA polymerase [Thermo Fisher Scientific], 5 µM biotin-14-dCTP, 5 µM each of normal dATP, dGTP and dTTP, 1.5 mM MgCl2, 25 nM of each primer, 1X PCR reaction buffer [Thermo Fisher Scientific] and sterile water up to 20 µl). The reaction mix was incubated under the following conditions in covered 96-well Multiply PCR plates: one cycle of 2 min at 94 °C, 30 cycles of 30 s at 94 °C, 30 s at 45 °C and 1 min at 72 °C, followed by a final extension of 5 min at 72 °C.
Pcr reaction buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
PCR reaction buffer is a solution used in the Polymerase Chain Reaction (PCR) process to provide the necessary components for DNA amplification. It contains a buffer system, salts, and other essential elements required for the optimal performance of the DNA polymerase enzyme during the PCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Mgcl2, by Thermo Fisher Scientific (3 mentions)
Dntps, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (2 mentions)
Taq polymerase, by Thermo Fisher Scientific (2 mentions)
Biotin 14 dctp, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dntp solution mix, by New England Biolabs (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Issr primers, by Metabion (1 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
Pgl3 enhancer vector, by Promega (1 mentions)
Neb cutsmart buffer, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Reverse transcriptase buffer, by Promega (1 mentions)
Random primer, by Promega (1 mentions)
10 protocols using pcr reaction buffer
1
Multiplex Tospovirus Detection and Identification
2
Quantification of Endothelial-Derived cfDNA
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Endothelial-derived cfDNA was quantified using the methylation status-specific CDH5 qPCR primers described above against a standard curve generated with bisulfite converted in-vitro unmethylated human DNA (Zymo Research) serially diluted 1 in 2. The 20 μL PCR reaction contained: 5 μL of the eluted bisulfite-converted cfDNA (0.667 mL of plasma equivalent), 1X PCR Reaction Buffer (Thermo Fisher Scientific), 0.2 mM dNTP Solution Mix (New England Biolabs), 0.15 U/μL Platinum Taq DNA Polymerase (Thermo Fisher Scientific), 2.5 μM Syto 9 (Thermo Fisher Scientific), 2.5 mM MgCl2 (Thermo Fisher Scientific), and 0.2 μM CDH5 forward and reverse each (Sigma-Aldrich). The qPCR started with 95°C for 3 min then for 45 cycles, 95°C for 10s, 62°C for 20s and 72°C for 30s (Thermo Fisher Scientific QuantStudio ViiA 7 Real-Time System). The quantification was expressed as ng of endothelial cfDNA per 1 mL of plasma. The relative proportion of the endothelial cfDNA to total cfDNA amount was calculated by:
3
Optimized ISSR-PCR Profiling of Acid Lime
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Using optimized ISSR–PCR reaction conditions (25 ng genomic DNA, 3.0 mM MgCl2, 2.5 µL (10 mM), 10× PCR reaction buffer (Thermo Fisher Scientific, Waltham, MA, USA), 0.4 µM Primer, 0.4 mM dNTPs, and 1.5 U Taq polymerase in a 25 µL reaction volume) and cycling conditions, as mentioned in the previous section (Section 2.2 ), 49 different ISSR oligos were used to screen the acid lime genomic DNA samples. All profiling experiments were repeated twice to check the reproducibility of PCR amplifications. Out of 49 oligos suggested by different researchers in their published papers [23 (link),45 (link),46 ], 21 oligos that provided crispy, multiple, scorable, and reproducible bands with our samples were selected for further ISSR profiling. The ISSR profiles generated by each of the 21 oligos were used to score the bands and for the creation of binary data matrix. Scoring of all polymorphic and monomorphic bands was performed. Scoring of the markers as 0, 1, or 9 was performed to indicate absence, presence, or failure of the PCR amplification (250–3200 bp) respectively [47 (link),48 ,49 (link)].
4
ISSR Primers for B-specific DNA Profiling
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For discrimination of B-specific DNA profiles we tested twelve ISSR primers (Metabion, Germany) with dinucleotide and trinucleotide repeats: (CAG)5, (CAA)5, (CAG)5GC, (CAG)4AC, (CAA)5GC, (CAA)4AC, (CA)6CG, (CA)8TA, (GA)6GC, (GA)8AC, (CA)7 and (GA)7. There were eight anchored primers with a dinucleotide tail attached to the 3’ end and four non-anchored ones. PCR were optimized according to [44 (link)]. Optimized conditions for a final volume of 20 μl were: PCR reaction buffer [750 mM Tris–HCl (pH 8.8 at 25°C), 200 mM (NH4)2SO4, 0.1% Tween 20] (Thermo Scientific) 2.50 mM MgCl2, 1mM dNTPs, 0.5μM primer, 1 U of DreamTaq DNA Polymerase (Thermo Scientific, Waltham, MA USA) and 20 ng of gDNA. The temperature profile was: initial denaturation at 94°C for 5 min, 45 cycles (94°C for 30 s; 58°C for 30 s; 72°C for 1.5 min) and a final extension at 72°C for 7 min. We used 200 μl microtubes in a Thermal Cycler 2720 (Applied Biosystems, Boston, CA, USA). Amplification products were separated by agarose gel electrophoresis. The DNA fragments were visualized with UV light using the Bio-Rad Gel Doc XR+ System and the results photo-documented using Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Inc., California, USA).
5
Quantifying Immune Receptor Expression in Ovarian Cancer
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RT-PCR was used to confirm the expression of TLR3, MDA5, RIG1, and PKR in the ID8-VegfA mouse ovarian cancer cell line and in the human ovarian carcinoma cell lines, A2780, COV362 and SKOV3. In addition, the same technique was used to determine expression of other TLRs in ID8-VegfA cells, and chemokine receptors in isolated DCs. Reactions were set up in a 25μl total volume, with each one using PCR Reaction Buffer, Mg+2, dNTPs, and Taq DNA Polymerase (all from Invitrogen; Grand Island, NY). The primers were designed using PrimerBLAST (NCBI) and were used at a concentration of 0.4 μM. For analysis, each reaction product was mixed with 10μl of 6x loading buffer (Invitrogen; Grand Island, NY), and resolved on a 1.5% agarose gel, run at 100V for thirty minutes in 1xTAE buffer, using the 100 bp DNA Ladder (Invitrogen; Grand Island, NY) for reference. The primer sequences for each gene of interest, together with the amplicon size are listed in Table 1 .
6
Sequence Analysis of Genomic Deletions
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For all cases, a PCR was designed to span the deletion breakpoint. PCR reactions were performed in a 25 μL final volume. This comprised 10 μmol of forward and reverse primer, 200 μM of each dNTP, 1× PCR reaction buffer (Invitrogen), 1.0 mM or 1.5 mM MgCl2 (Invitrogen), 1 unit of Taq Polymerase (Invitrogen) and 50 ng of genomic DNA made up to 25 μL with nuclease‐free water. Thermocycling conditions consisted of a denaturation step at 96°C for 3 min followed by 30–35 amplification cycles comprising 92°C for 30 s, 60–65°C for 30 seconds and 72°C for 30 s before a final extension step at 72°C for 10 min. For all cases, the long‐range PCR amplicon was directly sequenced using internal sequencing primers.
Sanger sequencing reactions were performed using BigDye Terminator v.3.1 and resolved on an ABI3130xl Genetic Analyser (Applied Biosytems) according to the manufacturer's instructions. Electropherograms were visualised using 4Peaks (https://nucleobytes.com/4peaks/ ).
Sanger sequencing reactions were performed using BigDye Terminator v.3.1 and resolved on an ABI3130xl Genetic Analyser (Applied Biosytems) according to the manufacturer's instructions. Electropherograms were visualised using 4Peaks (
7
Cloning and Validating MERTK Promoter Fragments
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Seven overlapping fragments (each approximately 1 kb in size) that spanned a 5.735 kb region (chr2 : 112,651,004-112,656,738) from –5187 bp to +548 bp of the MERTK transcription start site were cloned using healthy control human DNA (see above) as a template. Primer pairs were designed using UCSC genome browser with the human reference genome (GRCh37/hg19) and were sourced from Integrated DNA technologies (Coralville, IA, USA) (Table 1 ). PCR reactions were carried out using 2 U recombinant Taq DNA polymerase recombinant (5 U/μl) (Invitrogen, CA, USA), 1× PCR reaction buffer (Invitrogen), 2 mM dNTPs, 1.5 mM MgCl2, 0.5μM forward and reverse primers and 100 ng of human DNA. PCR cycling conditions were as follows: 1 cycle of 95°C for 3 minutes; 35 cycles of 95°C for 30 seconds, annealing temperature (Individual Ta for each amplicon outlined in Table 1 ) for 30 seconds, 72°C for 2 minutes; followed by 1 cycle of 72°C for 10 minutes.
PCR amplicons and pGL3 enhancer vector (Promega, USA) were digested with appropriate restriction enzymes (NEB, USA) in 10× NEB cutsmart buffer (NEB). Each PCR amplicon (50 ng) was ligated into the reporter vector (50 ng) using 400 Units T4 DNA ligase (400 U/μl) (NEB). Plasmids were sequenced using Sanger sequencing (AGRF, VIC, AUS) to validate DNA sequence of cloned amplicons within pGL3 enhancer vector.
PCR amplicons and pGL3 enhancer vector (Promega, USA) were digested with appropriate restriction enzymes (NEB, USA) in 10× NEB cutsmart buffer (NEB). Each PCR amplicon (50 ng) was ligated into the reporter vector (50 ng) using 400 Units T4 DNA ligase (400 U/μl) (NEB). Plasmids were sequenced using Sanger sequencing (AGRF, VIC, AUS) to validate DNA sequence of cloned amplicons within pGL3 enhancer vector.
8
RNA Extraction and cDNA Synthesis Protocol
9
Leishmania rRNA ITS-1 Detection via PCR
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Total genomic DNA was extracted from tissues of the liver, spleen, and bone marrow of 8 and 16 weeksinfected BALB/c mice and hamsters using a genomic DNA purification kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's instructions. Parasite DNA was detected by amplification of Leishmania rRNA ITS-1 using the LeF/LeR primers (Spanakos et al. 2008) . The PCR reaction mixture contained template DNA, 1×PCR reaction buffer (Invitrogen, Carlsbad, CA, USA), 4 mM MgCl2 (Invitrogen, Carlsbad, CA, USA), 0.6 μM of each primer (Invitrogen, Carlsbad, CA, USA), 0.8 mM of each dNTPs (Invitrogen, Carlsbad, CA, USA), and 1U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). Amplification was performed in TPersonal Combi Thermocycler (Biometra, Göttingen, Germany) using a step of initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min, extension at 72 °C for 2 min, and a final extension step at 72 °C for 5 min. Amplified products were run on 1.2% agarose gels (Amresco, Atlanta, GA, USA) containing ethidium bromide.
10
Microbial Community DNA Extraction and 16S Amplification
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Community DNA was extracted from frozen sediment samples using the UltraClean Soil DNA Isolation Kit (Mo BIO; Carlsbad, CA), according to the manufacturer's instructions. The genomic DNA extracted was used for the PCR amplification of partial bacterial 16S rRNA genes , using the specific primer 341f-GC (Muyzer et al., 1993) and 518R (Sánchez et al., 2007) . Each PCR mixture contained: 5 μl of 10× PCR reaction buffer (Invitrogen), 2.5 μl of 50 mM MgCl 2 , 1 μl of a 10 mM dNTP mixture, 1 μl of 10 μM (each) primer, 1 unit of Taq polymerase, at least 60 ng of the extracted DNA, and sterile MilliQ water up to 50 μl. The PCR programme for bacteria was: 94ºC for 5 min, 65ºC for 1 min, 72ºC for 3 min, and 9 touchdown cycles of: 94ºC for 1 min, 65ºC (with a decreas e of 1ºC in each cycle) for 1 min, 72ºC for 3 min, followed by 20 cycles of: 94ºC for 1 min, 55ºC for 1 min, and 72ºC for 3 min (Muyzer et al., 1993) . During the final cycle the extension step was increased to 30 min to minimise double band formation (Janse et al., 2004) and eliminate heteroduplexes; 5 μl of PCR products were used as templates for a 5-cycle reamplification (65°C and 55°C annealing temperature for bacteria and eukaryotes, respectively), using fresh reaction mixture, as described by Thompson et al. (2002) .
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