Various β-lactamase genes and the genes that encode DNA gyrAse (gyrA) and topoisomerase IV (parC) were detected using two separate multiplex PCR assays. Primers gyrA-F (5'-GTGTGCTTTATGCCATGAG-3') and gyrA-R (5’-GGTTTCCTTTTCCAGGTC-3') (24 (link)) were used to amplify 287 bp of the fluoroquinolone resistance-determining region of the gyrA gene. Primers parC-F (5'- CATCGTCTACGCCATGAG-3') and parC-R (5'-AGCAGCACCTCGGAATAG-3') (24 (link)) were used to amplify 267 bp of the fluoroquinolone resistance-determining region of parC. PCR amplification was performed in a 50 μL mixture, containing 1 × PCR buffer, 2.5 mM MgCl2, 0.2 mM mix of deoxynucleotide triphosphates (Fermentas), 10 pmol of each primer, 1 U of Taq DNA Polymerase (Fermentase), and 150 ng of the DNA template. Amplification of the target regions was performed in 35 cycles consisting of initial heat activation at 95 °C for 6 min, denaturation at 95 °C for 45 s, annealing at 51°C for 45 s and elongation at 72 °C for 1 min, with a final elongation at 72°C for 7 min. List of primers used for detection of various β-lactamase genes is shown in Table 2 18. The PCR reaction was performed in a total volume of 25 µL, containing 2 µL of DNA template, 1.4 mM MgCl2, 150 µM of each dNTP (Fermentas), 0.3 µM of each primers and 1 U Taq DNA Polymerase (Fermentas). The cycling parameters used were as previously described (18 (link)).
Deoxynucleotide triphosphate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Deoxynucleotide triphosphate is a key chemical building block used in DNA synthesis and amplification. It serves as a fundamental component in various molecular biology techniques, including PCR, DNA sequencing, and DNA cloning.
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Lab products found in correlation
Taq dna polymerase, by Thermo Fisher Scientific (5 mentions)
Moloney murine leukemia virus m mulv reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Oligo dt primer, by Roche (3 mentions)
Platinum sybr green qpcr supermix udg kit, by Thermo Fisher Scientific (3 mentions)
Nucleospin, by Macherey-Nagel (3 mentions)
Lightcycler system, by Roche (3 mentions)
Mgcl2, by Thermo Fisher Scientific (3 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Pcr buffer, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis reaction mixture, by Bio-Rad (2 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (2 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (2 mentions)
Para nitrophenyl phosphate, by Merck Group (1 mentions)
Mccoy s medium, by Merck Group (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Alizarin red, by Merck Group (1 mentions)
17β estradiol, by Merck Group (1 mentions)
23 protocols using deoxynucleotide triphosphate
1
Multiplex PCR for Detecting Resistance Genes
2
PCR-based Detection of mec C Gene
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Uniplex PCR based testing of mecC gene (a homolog of mecA gene) was carried out for all the staphylococcal isolates (Table-1 ) [22 (link)]. PCR assay was done in a 15 μL reaction volume containing 1X PCR ready master mix (0.025U DNA Taq polymerase in reaction buffer, 2 mM MgCl2, and 200 mM deoxynucleotide triphosphates [Fermentas, Glen Burnie, MD, USA]), 0.5 μM of mecC specific primers and 50 ng of extracted DNA. The PCR cycling conditions comprised an initial denaturation step at 95°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 50°Cfor 30 s, extension at 72°C for 30 s, and final extension step at 72°C for 4 min.
3
Cell Proliferation and Differentiation Assays
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McCoy’s medium, fetal bovine serum, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 17β-estradiol, alizarin red, para-nitrophenyl phosphate, diethanolamine, ascorbic acid, TRI reagent, custom-prepared oligonucleotides, lipopolysaccharide (LPS), and dexamethasone were obtained from Sigma (St. Louis, MO, USA). Penicillin, streptomycin, and magnesium chloride were from Hi-media (Mumbai, India). MMLV reverse transcriptase, deoxynucleotide triphosphates, and Taq DNA polymerase were purchased from MBI Fermentas (Amherst, NY, USA). All other reagents and chemicals used were of molecular biology grade.
4
RNA Isolation and qPCR for Organoids
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According to the manufacturer’s protocol, the total RNA was isolated from three pooled wells for organoids using Bioline ISOLATE II RNA Mini Kit (GC Biotech, Alphen aan den Rijn, The Netherlands). cDNA was synthesized by using deoxynucleotide triphosphates (Thermo Fisher Scientific, Waltham, MA, USA), random primers (Promega, Leiden, The Netherlands), Oligo dT primers (Sigma-Aldrich Chemie NV, Zwijndrecht, The Netherlands), Revertaid, and Ribolock (both Thermo Fisher Scientific). Quantitative PCR was performed with SensiFAST SYBR No-ROX (GC Biotech) and a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories B.V., Lunteren, The Netherlands). Analysis was performed with LinRegPCR software. For normalization, reference genes were used that were previously selected with GeNorm software. Primers were obtained from Sigma. Table S1 shows the primer sequences.
5
Preparation of High-Fidelity DNA Polymerase Reagents
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Enzymes, deoxynucleotide triphosphates and Phusion™ high-fidelity DNA-polymerase was obtained from ThermoFisher Scientific (Vienna, Austria). 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and hemin were purchased from Sigma-Aldrich (Vienna, Austria). Difco™ yeast nitrogen base w/o amino acids (YNB), Difco™ yeast nitrogen base w/o amino acids and ammonia sulfate (YNB2), Bacto™ tryptone and Bacto™ yeast extract were purchased from Becton–Dickinson (Vienna, Austria). Zeocin™ was purchased from InvivoGen (Toulouse, France) via Eubio (Vienna, Austria).
6
ATXN3 Gene Amplification Protocol
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The CAG tract and flanking region of the ATXN3 gene was PCR amplified by using a pair of primers designed and validated in our laboratory (Table 1). The design was based on the annotated reference sequence for the ATXN3 gene at the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/gene, accession number NG_ 008198.2), using the online Primer-BLAST design tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast).
Briefly, each PCR reaction (50 mL) was prepared on ice, using the following components: 10 mL of 5X Phusion GC Buffer (Thermo Fisher Scientific, Waltham, MA), 1 mL of 10 mmol/L deoxynucleotide triphosphates (Thermo Fisher Scientific), forward and reverse primers (Table 1) with a final concentration of 0.5 mmol/L each, 10 to 20 ng of DNA template, 2.5 mL (5%) of dimethyl sulfoxide (Thermo Fisher Scientific), and 0.5 mL (one unit) of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). The PCR amplification was performed in a Veriti 96-Well Thermal Cycler (Thermo Fisher Scientific) with the following protocol: 1 cycle at 98 C for 1 minute (initial denaturation) and 35 cycles at 98 C for 30 seconds (denaturation), 60 C for 10 seconds (annealing), and 72 C for 30 seconds (extension), with a final extension at 72 C for 10 minutes.
Briefly, each PCR reaction (50 mL) was prepared on ice, using the following components: 10 mL of 5X Phusion GC Buffer (Thermo Fisher Scientific, Waltham, MA), 1 mL of 10 mmol/L deoxynucleotide triphosphates (Thermo Fisher Scientific), forward and reverse primers (Table 1) with a final concentration of 0.5 mmol/L each, 10 to 20 ng of DNA template, 2.5 mL (5%) of dimethyl sulfoxide (Thermo Fisher Scientific), and 0.5 mL (one unit) of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). The PCR amplification was performed in a Veriti 96-Well Thermal Cycler (Thermo Fisher Scientific) with the following protocol: 1 cycle at 98 C for 1 minute (initial denaturation) and 35 cycles at 98 C for 30 seconds (denaturation), 60 C for 10 seconds (annealing), and 72 C for 30 seconds (extension), with a final extension at 72 C for 10 minutes.
7
T7 Bacteriophage Propagation and Quantification
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The T7 genomic DNA was purchased from Boca Scientific. The chi6 short DNA (Integrated DNA Technologies) was added at a concentration of 3 µM to prevent the degradation of the linear T7 DNA (57 (link)). dNTPs (deoxynucleotide triphosphates) (Invitrogen) were added to a concentration of 0.1 mM each to enable genome replication, as described before (44 (link)). The PEG8000 (Sigma Aldrich) concentration was increased from 2% (2.5 mM) to 3.5% (4.3 mM) to emulate molecular crowding (43 (link)). Bacteriophages were counted by the standard plaque-forming assay using the E. coli strain B for T7. Cells were grown in Luria–Bertani (LB) broth at 37°C. The plates were prepared as follows: each sample was added to a solution composed of 5 ml of 0.6% liquid LB-agar (45°C) and 50 µl of cell culture, poured on a 1.1% solid LB-agar plate. Plates were incubated at 37°C, and plaques were counted after 6 h.
8
Colon Tissue RNA Extraction and qPCR Analysis
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Colon tissue was homogenized using a Precellys 24 tissue homogenizer and Lysing Matrix E tubes (MP Biomedicals) containing RLT buffer (QIAGEN) with β-mercaptoethanol (MilliporeSigma). This was followed by isolation of total RNA using the RNeasy Mini Kit (QIAGEN) as per the manufacturer’s instructions. Next, reverse transcription of total RNA was performed with deoxynucleotide triphosphates, Oligo(dT)20 Primers, RNaseOUT, and SuperScript IV reverse transcriptase (all Invitrogen, Thermo Fisher Scientific) as per the manufacturer’s instructions. Real-time polymerase chain reaction was carried out using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) on an Applied Biosystems 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific). Next, the expression of the genes of interest was normalized against that of housekeeping gene 18S ribosomal RNA (rRNA). The following primers were used: 18S rRNA, 5′-CATTCGAACGTCTGCCCTATC-3′ (Fw) and 5′-CCTGTGCCTTCCTTGGA-3′ (Rev); Tff3, Mm_Tff3_1_SG QuantiTect Primer Assay; cMyc, Mm_Myc_1_SG QuantiTect Primer Assay; Tpi1, Mm_Tpi1_1_SG QuantiTect Primer Assay; Pten, Mm_Pten_1_SG QuantiTect Primer Assay; and Hk2, Mm_Hk2_1_SG QuantiTect Primer Assay (QIAGEN).
9
Estrogen Signaling Modulation in MCF7 Cells
10
Quantification of Gene Expression in Ovarian Tissue
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