First, genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen). The primers for the detection of a 1,176 bp fragment of the PMI gene were 5′-GCACTCGAGCATGCAAAAACTCATTAACTCAG-3′ and 5′-GCACTCGAGCTCTTACAGCTTGT TGTAAAC-3′. The primers for a 386 bp fragment of the Rs-AFP1-SP1-1 were: 5′-CAGGCTTCACAATGGCTAAGT-3′ and 5′-CGCGCTATATTTTGTTTTCTATC-3′. PCR amplifications from genomic DNA were done using the PCR super mix (Invitrogen) following the manufacturer´s instructions. In other cases the Extract-N-Amp Plant PCR Kits (Sigma-Aldrich) was used as described according to the manufacturer´s instructions. Amplifications were performed in a thermocycler (Eppendorf). PCR products were analyzed by electrophoresis in 1 or 2% agarose gels.
Pcr supermix
Manufactured by Thermo Fisher Scientific
Sourced in United States
PCR SuperMix is a ready-to-use solution that contains all the necessary components for performing polymerase chain reaction (PCR) experiments, including a thermostable DNA polymerase, dNTPs, and buffer. It is designed to simplify PCR setup and improve consistency of results.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (3 mentions)
Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Dneasy plant mini kit, by Qiagen (2 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Flash gels, by Lonza (2 mentions)
Tae buffer, by New England Biolabs (2 mentions)
Hifi hotstart readymix, by Roche (2 mentions)
Ampicillin, by Merck Group (2 mentions)
Dichloromethane, by Merck Group (2 mentions)
Gentamicin, by Merck Group (2 mentions)
Erythromycin, by Merck Group (2 mentions)
Thermocycler, by Analytik Jena (2 mentions)
Tetra primer, by Merck Group (2 mentions)
Wpa lightwave 2, by Harvard Bioscience (2 mentions)
Iq5 rt pcr detection system, by Bio-Rad (2 mentions)
56 protocols using pcr supermix
1
Genetic Marker Detection Protocol
2
Reverse Transcription and qPCR Analysis
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1 μg of total RNA was reverse-transcribed using QuantiMir RT Kit (System Biosciences). The cDNA was diluted (100-fold) and amplified using PCR Supermix (Invitrogen) or iTaq SYBR Green Supermix with Rox (Bio-Rad) on GeneAmp PCR System 9700 or on ABI 7300 Real Time PCR system.
3
Evaluating Wnt Pathway Modulation
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P19 cells or HCT-116 cells were treated with different concentration of apigenin or carrier reagent (DMSO) in culture medium (HCT-116 cells) or in Wnt-3a-conditioned media (P19 cells) for the indicated time. Total RNAs were isolated from treated cells by using the Trizol reagent (Invitrogen) according to the company’s protocol. 5 μg of RNAs were used for the synthesis of cDNAs by using a cDNA synthesis kit (Invitrogen) and the resulting cDNAs were used as the template for the amplification of Wnt target genes or GAPDH gene by using the PCR SuperMix (Invitrogen). Gene-specific primers used were: mouse Axin2 (amplicon, 237 bp), forward, 5′-GTTAGTGACTCTCCTTCCAGATCC-3′, reverse, 5′-GAGTGTAAAGACTTGGTCCACCTG-3′, human Axin2 (amplicon, 237 bp), forward, 5′-GTTGGTGACTTGCCTCCCGGACCC-3′, reverse, 5′-GAGTGTAAGGACTTGGTCCACCGG-3′, human and mouse GAPDH (amplicon, 539 bp), forward, 5′-CGTATTGGGCGCCTGGTCACC-3′, reverse, 5′-GAGGGGCCATCCACAGTCTTC-3′, human c-Myc (amplicon, 505 bp), forward, 5′-CCAGGACTGTATGTGGAGCGG-3′, reverse, 5′-CTTGAGGACCAGTGGGCTGTG-3′, mouse T (amplicon, 371 bp), forward, 5′-GTGACCAAGAACGGCAGGAGGATG-3′, reverse, 5′-AAGCAGTGGCTGGTGATCATGCG-3′, and human and mouse cyclin D1 (amplicon, 257 bp), forward, 5′-ATGGAACACCAGCTCCTGTGCTGCG-3′, reverse, 5′-TCCAGGTAGTTCATGGCCAGCGGG-3′.
4
RNA Isolation and Real-Time PCR Analysis
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Renal tissues were homogenized, sonicated, and total RNA was isolated using Ambion kit (cat.no.12183018A, PureLink TM RNA Mini Kit, Invitrogen, Life Technologies, USA) for the biological and technical triplicates and 0.5 μg RNA was used to synthesized cDNA according to the manufacturer’s instructions (part no.4368813, High Capacity cDNA Revert Transcriptase Kit, Applied Biosystems Inc., USA). It was amplified under the following PCR conditions: 94°C for five minutes, 30 cycles of PCR (94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec) 72°C for seven minutes and then at 4°C using Master Mix (cat.no.10572-063, PCR Supermix, Invitrogen, USA). Samples were electrophoresed at 2% agarose gel. For a standard reaction in real-time PCR, cDNA in 1:10 dilution was used as a PCR template. cDNA was mixed together with 2.5 nM of each primer in a final volume of 25 μl. Real-time PCR was performed using LightCycler TaqMan Master Mix (Roche, Germany) through manufacturer’s guidelines. An endogenous ‘housekeeping’ gene, 18S rRNA was quantified and used to normalize the results. All the genes were compared with control (normal adult mice) and relative genes expressions were quantified through 2^−ΔΔCt method. The probe sequences are given in Supplemental Table 1 .
5
Validating Soybean Transgenic Insertions
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Primers were designed to generate an amplicon that contains a small portion of 5′ T-DNA end and 5′ flanking region or 3′ T-DNA end and 3′ flanking region. Primer sets of 35SproR/Gm11R, Gm03F/LB3, and LB3/GM14R were used for the insertion loci validation of soybean transgenic lines of 14A, 17B, and 16C, respectively (Supplementary Table S1 ). Untransformed soybean genomic DNA and distilled water were used as negative controls in PCR reactions. PCR was performed using genomic DNA of three transgenic plants and PCR SuperMix (Invitrogen, (Thermo Fisher Scientific) according to the manufacturer’s instructions. The PCR program was as follows: 94 °C (4 min); 28 cycles of and 94 °C (30 Re: revised manuscript submitted; one query from editor sec), 53 °C (30 s), and 68 °C (1 min); and then 72 °C (7 min). Amplified products were analyzed on 1% agarose gels.
6
Genotyping STING N153S Mutation in Mice
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Toe clips were digested with proteinase K, and DNA was isolated with the Qiagen kit per manufacturer’s protocol. Two separate PCR reactions were performed for genotyping: a WT allele PCR and a STING N153S mutant allele PCR. WT primer (5′-GTCTGTGAAGAAAAGAAGTTAAA-3′) or STING N153S forward primer (5′-GTCTGTGAAGAAAAGAAGTTAAC-3′) and reverse primer (5′-GTGATTTTATGTACCCTGGG-3′) recognizing sequences in exon 5 of Tmem173 (STING) were added to PCR SuperMix (Invitrogen) at a final concentration of 0.4 µM. PCR was performed with an initial denaturation (94°C, 2 min), followed by 34 cycles of denaturation (94°C, 30 s), annealing (47°C, 45 s), and extension (72°C, 1 min). Mice positive for the STING N153S mutation were identified by the appearance of a 258-bp band by agarose gel electrophoresis.
7
Detecting Phlebovirus RNA via RT-PCR
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The RNA was extracted from supernatant aliquots by using the QIAamp viral RNA kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s protocol. Phlebovirus detection was performed amplifying Phlebovirus RNA-dependent RNA polymerase gene on a portion of L segment using degenerated primers [37 (link)]. The RT- and nested-PCR were performed using Super script One step RT-PCR System Kit (Invitrogen, Gaithersburg, MD) and PCR SuperMix (Invitrogen) respectively, according to the manufacture’s recommendations. The PCR conditions were those previously described [37 (link)]. PCR products were analyzed in a 2 % TAE agarose electrophoresis gel.
8
Isolation and Characterization of Alkaline Protease Producers
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Twelve soil bacterial isolates (D2, D9, D10, D14, D26, D30, D35, D40, D42, D44, D46, and D48) showing the maximum alkaline protease productivity were used for amplification of the 16S rRNA gene. PCR was done directly from the bacterial glycerol stock according to Macrogen. Primers F 5′AGA-GTTTGATCCTGGCTCAG-3′ and R 5′-GGTTACCTTGTTACGACTT-3′ were used for PCR amplification of the 16S rRNA gene (Srinivasan et al., 2015 ). Isolate D9 with high protease activity was used for amplification of alkaline protease gene AKD9. Alkaline protease primers F 5′ CATATGTTTGGGTACTCTATGG-3′ and R 5′ GGATCCTTATTGGCCGGGAACGGAA-3′ were used (Sadeghi et al., 2009 ). PCR was performed according to the manufacture of PCR SuperMix Invitrogen™. The PCR product containing alkaline protease gene was extracted from 0.8% agarose gel using QIAquick Gel Extraction Kit (Qiagen), and inserted into TA Cloning® Kit, with pCR™2.1 Vector, Invitrogen. The recombinant plasmid was transformed into DH5α E. coli cells.
9
Semiquantitative RT-PCR for Rat KCNQ2 and KCNQ3
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To detect the expression of rat KCNQ2 and KCNQ3 mRNAs in different types of cells including GH3, R1220 cells, a semiquantitative RT-PCR assay was performed. Total RNA samples were extracted from mHippoE-14 cells with TRIzol reagent (Invitrogen) and reverse-transcribed into complementary DNA using Superscript II reverse-transcriptase (Invitrogen). The sequences of forward and reverse oligonucleotide primers used for KCNQ2 were 5′-CCCTGAAAGTCCAAGAGCAG-3′ and 5′-AGGCCCCATAGGTTTGAGTT-3′, respectively, while those for KCNQ3 were 5′-GTGGCTTCAGCATCTCACAA-3′ and 5′-CTTGTTGGAAGGGGTCCATA-3′, respectively. Amplication of KCNQ2 or KCNQ3 was made using PCR SuperMix from Invitrogen under the following conditions: 35 cycles composed of 30 sec denaturation at 95 °C. 30 sec primer annealing at 62 °C, 1 min extension at 72 °C, and followed by 72 °C for the final extension for 2 min. PCR products were analyzed on 1.5% (v/v) agarose gel containing ethidium bromide and then visualized under ultraviolet light. Optical densities of DNA bands were scanned and quantified by AlphaImager 2200 (ProteinSimple; Santa Clara, CA, USA).
10
Molecular Diagnosis of Leishmania Infection
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End-point PCR targeting the 120-bp conserved region of the Leishmania kDNA minicircles {[25 (link)] #253} was performed as described {[29 (link)] #264}, employing primers 5′-GGG (G/T)AGGGGCGTTCT(G/C)CGAA-3′ and 5′-(G/C)(G/C)(G/C)(A/T)CTAT(A/T)TTACACCAACCC C-3′. The second end-point PCR assay targeted amplification of the segment containing ITS 1, 2 and the 5.8S region, within the rRNA gene locus {[24 (link)] #252}. Primers employed were IR1 (5′-GCTGTAGGTGAACCTGCAGCAGCTGGATCATT-3′) and IR2 (5′-GCGGGTAGTCCTGCCAAACACTCAGGTCTG-3′). Reactions were performed in a final volume of 20 μl, with 50 ng of DNA, 0.2 μM of each oligonucleotide and PCR supermix (Invitrogen), following manufacturer’s instructions. PCR conditions were as follows: denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 60 sec, 55°C for 60 sec and 94°C for 120 sec with a final extension of 72°C for 10 min. The amplification reactions were analyzed by agarose gel electrophoresis, followed by silver or ethidium bromide staining. DNA from the reference L. infantum strain was used as positive control for PCR reactions.
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