For the 3′ UTR RACE, reverse transcription was realized with an oligo-dT adapter primer: (GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTTTTTTTTTTTTTTTT) as described (9 (link)). PCRs were performed in a final volume of 15 μl with 1 μl of RT product, 3 μl of reverse primer at 20 μM and 1 μl of primer at 20 μM, 7.5 μl 2× PCR MasterMix (ThermoScientific). The PCRcycling conditions were 94°C for 5 min, followed by 5 cycles at 94°C for 20 s and 72°C for 50 s, then 5 cycles at 94°C for 20 s and 70°C for 30 s and 72°C for 20 s and 25 cycles at 94°C for 20 s and 68°C for 20 s and 72°C for 30 s, followed by a final extension at 72°C for 7 min. The nested PCRs were realized in a final volume of 15 μl with 1 μl of primary PCR product, 1 μl of reverse and/or forward primers 20 μM, 7.5 μl 2× PCR MasterMix (Thermo Scientific).The PCR cycling conditions were 94°C for 5 min, followed by 35 cycles at 94°C for 20 s and 60°C for 20 s and 72°C for 30 s, followed by a final extension at 72°C for 7 min. Nested PCR products were purified using a NucleoSpin Gel and PCR Clean-Up column (Macherey-Nagel).Fragments were cloned into TOPO-TA vector with a TOPO-TA Cloning kit (LifeTechnologies).Cleavage sites were determined by Sanger sequencing of at least 10 Individual clones.
Pcr master mix
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PCR Master Mix is a pre-optimized solution for performing polymerase chain reaction (PCR) experiments. It contains essential components for DNA amplification, including DNA polymerase, nucleotides, and buffers, in a ready-to-use format.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (29 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (21 mentions)
Trizol, by Thermo Fisher Scientific (19 mentions)
Rneasy mini kit, by Qiagen (16 mentions)
Rneasy micro kit, by Qiagen (12 mentions)
Primescript rt master mix, by Takara Bio (12 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (10 mentions)
Applied biosystems 7500 fast real time pcr system, by Thermo Fisher Scientific (8 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (6 mentions)
T4 dna ligase, by Thermo Fisher Scientific (6 mentions)
Taqman probe, by Thermo Fisher Scientific (6 mentions)
Tri reagent, by Merck Group (5 mentions)
Sybr green, by Thermo Fisher Scientific (5 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (5 mentions)
Ethidium bromide, by Merck Group (5 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Taq polymerase, by Thermo Fisher Scientific (5 mentions)
Taqman primer, by Thermo Fisher Scientific (4 mentions)
Superscript 3, by Thermo Fisher Scientific (4 mentions)
313 protocols using pcr master mix
1
3' UTR RACE Protocol with Sanger Sequencing
2
Multiplex PCR for Eight E. coli PAIs
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Eight PAIs in E. coli isolates were investigated in this study using two multiplex PCRs (1 and 2) according to Sabaté et al [56 (link)]. Three PAI markers were targeted (PAI III536, PAI IV536, and PAI IICFT073) in the first reaction, yielding 200, 300, and 400 bp, respectively; the reaction mixture measured 20 μL and contained 3 μL of DNA template, 10 μL of 2X PCR master mix (Thermo scientific, Waltham, MA, USA), 6 μL of deionized water, and 1 μL of each of the six primers. The second multiplex PCR targeted five PAI markers (PAI IIJ96, PAI I536, PAI II536, PAI ICFT073, and PAI IJ96), yielding 2300, 1800, 1000, 930, and 400 bp fragments, respectively. The PCR reaction was performed in a total volume of 20 μL containing 3 μL of DNA template, 10 μL of 2X PCR master mix (Thermo scientific, Waltham, MA, USA), 2 μL of deionized water, and 1 μL of each primer. The primer sequences and expected sizes of amplicons for each PCR assay are given in Table 7 . Both multiplex PCRs were performed under the following conditions: 94 °C for 5 min; 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min; and a final extension at 72 °C for 10 min.
3
Site-Directed Mutagenesis via PCR-Based Methods
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For site-directed mutagenesis based on PCR using a pair of complementary or partially complementary primers, DNA templates have to be digested with a restriction enzyme before transformation assays. Briefly, PCR was performed in a 25 μL reaction mixture containing 12.5 μL PCR master mix (Thermo Scientific/NEB/Vazyme Co), 200 ng plasmids and 1 μL 25 μM forward and reverse primers. When PCR was finished, PCR products were digested for 1 h under 10 U Dpn I at 37℃. A transformation assay was performed using 3 μL digestion samples as described for the SMLP method. For the site-directed mutagenesis based on PCR using a pair of inverse primers, PCR was performed in a 25 μL reaction mixture containing 12.5 μL PCR master mix (Thermo Scientific /NEB /Vazyme Co), 0.5 ng plasmids and 1 μL 25 μM forward and reverse primers. PCR products were purified with a DNA purification kit (CWBiol) and were then phosphorylated using T4 polynucleotide kinase (NEB) according to the manufacturer’s manual. DNA ligation was performed in a 20 μL reaction mixture containing 5 μL phosphorylated samples and 5 U T4 DNA ligase (Thermo Scientific). A transformation assay was performed using 3 μL ligation samples as described for the SMLP method.
4
HEV Genotyping by RT-PCR and Sequencing
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For HEV genotyping, an RT-PCR with two primer sets was used to amplify the 5’ half of the VP1region of the viral genome as described previously[12 (link), 25 (link)]. Briefly, in the first round, a one-step RT-PCR was performed in 25 μl reaction mixture containing 20 pmol of the outer primers (EV-F; 5’- GYDGARACNGGVCACACRTC-3’ and EV-R; 5’ CTMATGAAHGGDATNGAYATBC-3’ ), 10 U AMV and 12.5 μl PCR master mix (Thermo Scientific). Two microliters from the first round was further amplified in 25μl reaction mixture containing 20 pmol of the inner two primers (ENTNES-F; 5’-GAYACWATGCARACVMGRCAYGT-3’ and ENTNES-R; 5’-GRGCAYTVCCYTCTGTCCA-3’ ) and 12.5 μl PCR master mix (Thermo Scientific). Negative and positive controls were used in each run. A band of 400 bp visualized on agarose gel electrophoresis indicated a positive result. PCR amplicons were purified using the NucleoSpin Gel and PCR Clean-up from Machery Nagel (Germany) before sending for commercial sequencing.
5
Gene Expression Analysis Protocol
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Once the experiments were concluded, cells were lysed and RNA was extracted using E.Z.N.A Total RNA Kit I according to the manufacturer’s protocol. Afterwards, RNA was purified using the Lucigen digestion kit following the supplier’s instructions. For the reverse transcription, the High-capacity cDNA Reverse Transcription Kit was used. The expression of IL-6, LCN2, CCL2, COL2A1, ACAN, SOX9, MMP13, and COLX was measured by RT-PCR using the MasterMix PCR (ThermoFisher Scientific) in combination with specific primers in a QuantStudio 3 (ThermoFisher) (Table 1 ). Relative quantitation was performed using the ΔΔCt Comparative Method. The results were expressed as fold change versus unstimulated control.
6
Chondrogenic Differentiation of ATDC5 Cells
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ATDC5 murine chondrogenic cell line was obtained from RIKEN Cell Bank (Koyadai, Tsukuba, Ibaraki, Japan). Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with Ham’s F-12, Fetal Bovine Serum (FBS), antibiotics Penicillin-Streptomycin, Transferrin, Sodium Selenite, L-Glutamine, Trypsin, TRI Reagent, FNT, 3′methoxy-4′nitroflavone (3-MF), mouse IL-1β, MTT reactive, chloric acid, SDS, and Griess reactives (phosphoric acid, sulphanilamide, and alpha-naftil-etil-enamide) were purchased from Sigma Aldrich (St. Louis, MO, USA). ZK164015 was acquired from Cienytech (Santiago de Compostela, Spain). Insulin (Actrapid 100 UI/ML) was kindly donated by the Pharmacy Service of the Clinical Hospital of Santiago de Compostela. E.Z.N.A. Total RNA Kit I was bought from Omega BIO-TEK (Norcross, GA, USA). Digestion kit was purchased from Lucigen (Middelton, WI, USA). High-capacity cDNA Reverse Transcription Kit was bought from Applied Biosystems, Life Technologies (Grand Island, NY, USA). MasterMix PCR was purchased from ThermoFisher Scientific (Waltham, MA, USA). IL-6, neutrophil gelatinase-associated lipocalin (LCN2), C-C motif chemokine 2 (CCL2), COL2A1, ACAN, type X collagen (COLX), SRY-Box transcription factor 9 (SOX9), and MMP13 primers were purchased from Sigma Aldrich. Cell culture plates were purchased from ThermoFisher Scientific.
7
RNA Extraction and Gene Expression Analysis
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RNA was isolated from the IFMs of 2–3-d-old flies. IFMs were removed from the bisected thoraces at 4° and immersed in Trizol (Sigma). Next, RNA was extracted with the help of Trizol (Sigma) as per the manufacturer’s protocol. cDNA was made using 1–2 µg of extracted RNA and a cDNA synthesis kit (Fermentas). The following primers were used: RP49 (FP: 5′-TTCTACCAGCTTCAAGATGAC-3′, RP: -5′-GTGTATTCCGACCACGTTACA-3′); upheld (up ) (FP: 5′-CTCGGGTGTCTCGGGCTCAC-3′ RP: 5′-CTCGAACGAGAAGATCTGGA-3′); and Opa1-like (FP: 5′-AACGGTGGAGCCAGTTCTCG-3′; RP: 5′-TGATCTCCGTCTGCAGCGTC-3′). Quantitative PCR was carried out using DyNAmoTM HS SYBR green mix (F-410L; Thermo Scientific). Fluorescence intensities were recorded and analyzed in an ABI Prism 7900HT sequence detection system (SDS 2.1; Applied Biosystems). The relative changes in gene expression were estimated after normalization to the expression of a housekeeping gene, RP49. For semiquantitative PCR, reactions were set up using the 2× PCR Mastermix (Fermentas) and PCR amplification was carried out using a Mastercycler Nexus (Eppendorf).
8
PCR Amplification and Purification
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Example 6
Fifty microliter aqueous mixtures were prepared that contained 25 μL of 2×PCR Master Mix (K1071; Fermentas Inc. (Glen Burnie, Md.)), 0.5 μM each of SEQ ID NOs:1 and 2, and 3 μL of each gel melt volume. These mixtures were placed on a thermocycler with the program: (a) 95° C. for 4 min, (b) 25 cycles (95° C. for 30 sec., 60° C. for 30 sec., 72° C. for 120 sec.), and (c) 72° C. for 5 min. Electrophoresis following DNA amplification showed PCR products corresponding to the expected sizes. Two-closely spaced bands were seen for each reaction: one band corresponded to the duplexed product and another band appearing at slightly higher MW corresponded to product duplexed only at the adaptor ends.
Example 7
Individual 100 μL aqueous reaction mixtures were prepared containing 25 μl of 2×PCR Master Mix, 2 μM final concentration of each SEQ ID NO:1-SO and SEQ ID NO:2-SO and 2 μL of templates (1 pmole) obtained from Example 6. The reaction mixtures were placed in a thermocycler set with the following thermocycling program: 15 cycles (95° C. for 30 sec., 60° C. for 30 sec., 72° C. for 3 min.). After completion of thermocycling, the reaction products were isolated by precipitation with polyethylene glycol, resuspended in TE buffer, and the concentrations measured by spectrophotometery.
9
PCR Amplification and Restriction Analysis of GH Gene
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Based on the published primer by [9 (link)], the following forward and reverse primers were used for PCR amplification of the exon 2 and exon 3 region of GH gene.
Forward ‘5’-CTCTGCCTGCCCTGGACT-3’
Reverse 5’-GGAGAAGCAGAAGGCAACC-3’
The 25 μl PCR reaction mixture consisted of 3 μl (90 n g) template DNA, 1 μl of each forward and reverse primer, 12.5 μl of 2 × PCR master mix (Fermentas) and 7.5 μl of deionized water (DNAse free water). The PCR protocol comprised of an initial denaturation for10 min at 94°C followed by 30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 54°C and extension for 1 min at 72°C and final extension at 72°C for 10 min. The PCR product was analyzed electrophoretically in 2% agarose gel. Restriction digestion of amplicon was conducted in a total volume of 30 μl reaction mixture having 10 × buffer tango 2 μl, PCR reaction mixture 10 μl, restriction enzyme HaeIII (10 units/μl) 1 μl and 17 μl nuclease free water. The digested products were separated on 2% agarose gel, and images were documented in a gel doc system (Bio-Rad USA) for analysis.
Forward ‘5’-CTCTGCCTGCCCTGGACT-3’
Reverse 5’-GGAGAAGCAGAAGGCAACC-3’
The 25 μl PCR reaction mixture consisted of 3 μl (90 n g) template DNA, 1 μl of each forward and reverse primer, 12.5 μl of 2 × PCR master mix (Fermentas) and 7.5 μl of deionized water (DNAse free water). The PCR protocol comprised of an initial denaturation for10 min at 94°C followed by 30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 54°C and extension for 1 min at 72°C and final extension at 72°C for 10 min. The PCR product was analyzed electrophoretically in 2% agarose gel. Restriction digestion of amplicon was conducted in a total volume of 30 μl reaction mixture having 10 × buffer tango 2 μl, PCR reaction mixture 10 μl, restriction enzyme HaeIII (10 units/μl) 1 μl and 17 μl nuclease free water. The digested products were separated on 2% agarose gel, and images were documented in a gel doc system (Bio-Rad USA) for analysis.
10
Microsatellite Loci Primer Validation
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Primers for microsatellite loci were identified and validated by Minárik et al. [19 (link)] (see Additional file 1 : Table S1). Prior to PCR amplification with fluorescently labelled primers and fragment analysis, standard PCR amplification for all 11 microsatellite assays was performed in order to eliminate individuals with degraded DNA displayed by a loss of specific primer binding sites or degradation of amplification targets.
In total, 20 μl of amplification mixture contained 10–20 ng of gDNA, 10 μl 2× PCR Master Mix (Fermentas, UAB, Vilnius, Lithuania) and 10 pmol of each of the primers (Additional file1 : Table S1). The conditions of PCR reactions were: 5 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C. The final step was 5 min at 72 °C. The amplified PCR products were visualised on 1 % agarose gels.
In total, 20 μl of amplification mixture contained 10–20 ng of gDNA, 10 μl 2× PCR Master Mix (Fermentas, UAB, Vilnius, Lithuania) and 10 pmol of each of the primers (Additional file
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