Mgcl2 solution

Manufactured by Thermo Fisher Scientific

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MgCl2 Solution is a laboratory reagent that contains magnesium chloride dissolved in water. It is a clear, colorless liquid with a neutral pH. The solution is commonly used as a source of magnesium ions in various chemical reactions and analytical procedures.

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MgCl2 (467 citations) 25 mM MgCl2 solution (2 citations)

5 protocols using mgcl2 solution

PCR Amplification and Sequence Analysis

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In a PCR reaction tube the following reactants were combined: 10X PCR buffer (Invitrogen, Burlington, ON), 50 mM MgCl₂ solution (Invitrogen, Burlington, ON), 5X TAQ DNA polymerase (Invitrogen, Burlington, ON), 10 mM deoxyribonucleotide triphosphates (dNTPs), 0.1 mM primer, 2.5 ng DNA, and water to a final volume of 50 μl. The reactions were then placed in a thermocycler under the following conditions: 94°C for 3 minutes, 94°C for 30 seconds, 56°C for 1 minute, and 72°C for 1 minute for 30 cycles. Sequences of PCR products were determined by Robarts Research Institute DNA Sequencing Facility in London, ON, and were analyzed using BioEdit v7.0.5.

Morrison E.A., Garner S., Echaubard P., Lesbarrères D., Kyle C.J, & Brunetti C.R. (2014). Complete genome analysis of a frog virus 3 (FV3) isolate and sequence comparison with isolates of differing levels of virulence. Virology Journal, 11, 46.

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RT-PCR Protocol for EMC-Orf1a Detection

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A 25 µL reaction was prepared containing 5 µL of RNA, as described previously [31 (link)]. Briefly, 12.5 µL of 2× reaction buffer from the Superscript III one-step RT-PCR system with Platinum Taq Polymerase (Invitrogen, Carlsbad, CA, USA), 1 µL of reverse transcriptase/Taq mixture from the kit, 0.4 µL of a 50 mM MgCl₂ solution (Invitrogen, Carlsbad, CA, USA), 1 μL each of 10 μM forward and reverse of EMC-Orf1a primers with 400 nM concentration in the final solution, as well as 0.5 μL of 10 μM EMC-Orf1a probe with 200 nM concentration in the final solution was used. Molecular grade deionized water was used to make the final volume to 25 μL. Thermal cycling involved reverse transcription at 55 °C for 20 min, followed by denaturation at 95 °C for 3 min, and then 45 cycles of denaturation and extension at 95 °C for 15 s, and 58 °C for 30 s.

Naeem A., Hamed M.E., Alghoribi M.F., Aljabr W., Alsaran H., Enani M.A, & Alosaimi B. (2020). Molecular Evolution and Structural Mapping of N-Terminal Domain in Spike Gene of Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Viruses, 12(5), 502.

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Nested PCR for Treponema Phylogroups

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The PCR analyses were performed at Denmark Technical University. Altogether 168 cytobrush samples had enough DNA for the analysis. An initial PCR step using a universal bacterial oligo pair encompassing most of the 16S rRNA gene [21 (link)] was followed by nested PCR analysis using oligos specific for the three DD Treponema phylogroups as described by Evans et al. [21 (link)] (Table 2). In all PCR assays, a 25 μL reaction mixture contained 1.25 U AmpliTaq DNA polymerase (Applied Biosystems, CA, USA), 1.5 mM (universal oligos) or 3 mM (group specific oligos) MgCl₂ Solution (Applied Biosystems, USA), 100 μM of each dNTP (Amersham Biosciences, NJ, USA), 0.2 μM of each specific oligo, and 1 μL of the template in PCR Buffer II (Applied Biosystems, USA). Thermal cycling was performed in a T3 thermocycler (Biometra, Göttingen, Germany) as described by Evans et al. [21 (link)]. In each assay, water served as a negative control, and genomic DNA from each of the three Treponema groups as positive control. PCR products were separated on a 2% E-gel (Invitrogen, Carlsbad, 92,008 CA, USA), and visualized by UV fluorescence.

Kontturi M., Junni R., Simojoki H., Malinen E., Seuna E., Klitgaard K., Kujala-Wirth M., Soveri T, & Pelkonen S. (2019). Bacterial species associated with interdigital phlegmon outbreaks in Finnish dairy herds. BMC Veterinary Research, 15, 44.

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General PCR Reagents and Kits

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AmpliTaq Gold DNA Polymerase with Buffer II and MgCl₂ solution for general PCR reactions were purchased from Applied Biosystems, Foster City, CA. Restriction enzymes and modification enzymes were purchased from New England BioLabs, Beverly, MA. TOPO TA Cloning Kit (with pCR2.1-TOPO vector pCRII-TOPO vector) with One Shot Chemically Competent E. coli, TOPO Shotgun Subcloning Kit and LipofectAMINE 2000 reagent were purchased from Invitrogen, Carlsbad, CA. Oligonucleotides for PCR based reactions were purchased from The Molecular Resource Facility UMDNJ, Newark, NJ. Cell culture media was purchased from Cellgro by Mediatech, Inc., Herndon, VA. Except Fetal bovine serum was purchased from Hyclone, UT. Insulin-Transferrin-Sodium Selenite media supplement was purchased from Sigma, St. Louis, MO.

Muniz L.C, & Molina C.A. (2016). The transcriptional repressor ICER binds to multiple loci throughout the genome. Biochemical and biophysical research communications, 478(3), 1462-1465.

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Molecular characterization of Coxiella burnetii

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Molecular characterization of Coxiella burnetii DNA was performed by MST assay as previously described (Di Domenico et al., 2014) . Briefly, each reaction consisted of 1 × PCR Buffer II (Applied Biosystems), 200 nM of each primer (Glazunova et al., 2005) , 200 μM dNTPs (Promega), 2.5 mM MgCl2 Solution (Applied Biosystems), 0.03 U/μL AmpliTaqGoldTM (Applied Biosystems), 5 μL of DNA and PCR grade water to a final volume of 50 μL. Amplification was performed in a GeneAmp PCR System 9700 (Applied Biosystems) under the following conditions: initial denaturation of 10 min at 95 °C, followed by 45 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 57 °C, and exten-sion for 30 s at 72 °C, with a final extension at 72 °C for 7 min. The reac-tion mix for the Cox56 and Cox57 spacers PCRs were modified as previously described by Di Domenico et al. ( 2014). PCR products were purified using the Expin™ PCR SV Kit (GeneAll) and sequenced by using BigDye Terminator v.3.1 (Applied Biosystems) and the 3130 XL Genetic Analyzer (Applied Biosystems).
Raw sequence data were as-sembled using DNAStar Navigator and the sequences were compared with those reported in the reference database available on the website http://ifr48.timone.univmrs.fr/mst/coxiella_burnetii/strains.html.

Galiero A., Fratini F., Cammà C., Di Domenico M., Curini V., Baronti I., Turchi B, & Cerri D. (2016). Occurrence of Coxiella burnetii in goat and ewe unpasteurized cheeses: Screening and genotyping. International journal of food microbiology, 237.

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