Each multiplex ligation mix included 5 nM individual MOLigo pair probes (Table 2 ) with 2.5 μL10X Hifi Taq DNA ligase reaction buffer, 0.5 μL Hifi Taq DNA ligase (New England BioLabs, Massachusetts, USA), 0.1 μL of 0.05 ng/μL IC cloned into the plasmid and 2.5 μL template DNA. The reaction was brought to a final volume of 25 μL with H2O. Ligation protocol in the thermocycler (DNA Engine Dyad, Bio-Rad, Foster City, CA, USA) was set to: 10 min of denaturation at 95 °C followed by 20 cycles of 30 s at 95 °C and 1 min at 59 °C. Ligation products were stored at 10 °C until the next step of the MOL-PCR reaction.
Dna engine dyad
Manufactured by Bio-Rad
Sourced in United States
The DNA Engine Dyad is a thermal cycler designed for PCR (Polymerase Chain Reaction) amplification of DNA samples. It features a dual-block configuration, allowing two separate experiments to be run simultaneously. The instrument provides precise temperature control and consistent heating and cooling rates to ensure accurate and reliable results during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Hifi taq dna ligase, by New England Biolabs (1 mentions)
Pop7 polymer, by Thermo Fisher Scientific (1 mentions)
Tetrad2 thermal cycler, by Bio-Rad (1 mentions)
Qiamp dna blood mini kit, by Qiagen (1 mentions)
Betaine, by Tokyo Chemical Industry (1 mentions)
Abi prism 310 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Annealing buffer, by Qiagen (1 mentions)
Streptavidin sepharose high performance bead, by GE Healthcare (1 mentions)
Pyromark pcr master mix, by Qiagen (1 mentions)
Coralload concentrate, by Qiagen (1 mentions)
Pyromark binding buffer, by Qiagen (1 mentions)
Pyromark wash buffer, by Qiagen (1 mentions)
Pyromark q24, by Qiagen (1 mentions)
Pyromark gold q24 reagent, by Qiagen (1 mentions)
Rnase free water, by Qiagen (1 mentions)
Protease inhibitor, by Roche (1 mentions)
7 protocols using dna engine dyad
1
Multiplex Ligation-Dependent Probe Amplification
2
Multiplex ligation-dependent probe amplification for DMD
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We analyzed all DMD cases for large deletions and large duplications using MLPA SALSA P034/P035 DMD kits (http://www.mrc-holland.com ) following the manufacturer’s instructions. In brief, denaturation, hybridization, ligation, and amplification steps were performed on a DNA Engine Dyad thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA). Finally, PCR amplification was performed using SALSA MLPA PCR primers labeled with the FAM dye. A mixture of 0.7 μl of PCR product, 0.2 μl of 600 LIZ GS size-standard, and 9.0 μl of Hi-Di formamide was incubated for 3 min at 86 °C and cooled at 4 °C for 2 min. The MLPA product mix was separated on a POP7 polymer (Applied Biosystems Inc., Life Technologies, Foster City, CA) at 60 °C with the setting of 1.6 kV for injection voltage, 18 s for injection time, 15 kV for run voltage, and 1800 s for run time.
3
Genotyping of Pharmacogenomic Variants
4
Chromosome-specific PCR and Sanger Sequencing
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PCR was performed on “DNA Engine Dyad” (Bio-Rad, The Netherlands) with 1.25 U of “SmarNGTaq” DNA-polymerase (Dialat Ltd., Moscow, Russia); 1× “Ampli” PCR-buffer (Dialat Ltd., Moscow, Russia); 1.2 M Betaine (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan); 2.5 mM MgCl2 solution (Dialat Ltd., Moscow, Russia); and dNTPs (0.2 mMdATP, 0.2 mMdTTP, 0.2 mMdCTP, and 0.2 mMdGTP; Dialat Ltd., Moscow, Russia). The primers were as follows: 0.4 mM Rep1F 5′- TCCCACCCTCAGGCTCCTC-3′; 0.4 mM Rep1R1seq 5′-AGTGCAGACCAGGGCGCCG-3′. The PCR conditions consisted of an initial “hot-start” at 95 °C for 3 min, followed by 40 cycles: 95 °C—30 s, 62 °C—5 s, and 72 °C—15 s. The PCR product was 351 bp for chromosome 4q35 and 345 bp for chromosome 10q26.
Sanger sequencing of PCR-products was performed by the “DNA-diagnostic laboratory” (Moscow, Russia) on ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). The obtained sequences were analyzed with FinchTV v.1.5 software.
Sanger sequencing of PCR-products was performed by the “DNA-diagnostic laboratory” (Moscow, Russia) on ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). The obtained sequences were analyzed with FinchTV v.1.5 software.
5
Quantifying Soil Fungal Abundance
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Relative abundance of fungal 18S rRNA gene copy numbers was examined in all 24-soil fractions from the same DNA extracts that were utilized for the sequence libraries. Quantitative PCR was carried out using primers described by Castro et al. [41 ] as detailed in Weber et al. [40 ] in an MJ Research DNA Engine DYAD. Gene copy number was quantified three times for each of the six replicate soil samples collected per soil interval. After log transformation, the Shapiro and Fligner Killeen diagnostic tests for this data had P-values of > 0.05 and thus were statistically examined for the effects of vegetation, depth and an interaction of the two using conventional split-plot ANOVAs as described above.
6
Targeted Bisulfite Pyrosequencing Assay
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A custom pyrosequencing assay was designed to target the top differentially methylated position from the BeadChip data. The assay was designed using PyroMark Assay Design software 2.0 (Qiagen). Ten nanograms of template bisulfite‐converted DNA was added to 1× PyroMark PCR Master Mix (Qiagen), 1× CoralLoad Concentrate (Qiagen), and 0.2 μM PCR primer set (Qiagen). The solution was made up to 25 μl with RNase‐free water (Qiagen) and was amplified in a thermocycler (DNA Engine Dyad; MJ Research) using polymerase chain reaction (PCR) conditions recommended by the manufacturer (Qiagen).
The remaining PCR product (10–20 μl) was agitated at 1,400 revolutions per minute for 10 minutes with 2 μl streptavidin–Sepharose High Performance Beads (GE Healthcare) and 40 μl PyroMark Binding Buffer (Qiagen) made up to 80 μl with Milli‐Q water. The samples were applied to the vacuum handset of the PyroMark Q24 workstation and washed with 70% ethanol, PyroMark denaturation solution, and PyroMark wash buffer (Qiagen) before being added to 0.3 μM sequencing primer diluted to 25 μl in annealing buffer (Qiagen). To allow the biotin‐labeled DNA strand to anneal to the primers, the solution was heated to 80°C for 2 minutes, then allowed to cool to room temperature for 5 minutes. Pyrosequencing reactions were performed using a PyroMark Q24 and PyroMark Gold Q24 Reagents (Qiagen).
The remaining PCR product (10–20 μl) was agitated at 1,400 revolutions per minute for 10 minutes with 2 μl streptavidin–Sepharose High Performance Beads (GE Healthcare) and 40 μl PyroMark Binding Buffer (Qiagen) made up to 80 μl with Milli‐Q water. The samples were applied to the vacuum handset of the PyroMark Q24 workstation and washed with 70% ethanol, PyroMark denaturation solution, and PyroMark wash buffer (Qiagen) before being added to 0.3 μM sequencing primer diluted to 25 μl in annealing buffer (Qiagen). To allow the biotin‐labeled DNA strand to anneal to the primers, the solution was heated to 80°C for 2 minutes, then allowed to cool to room temperature for 5 minutes. Pyrosequencing reactions were performed using a PyroMark Q24 and PyroMark Gold Q24 Reagents (Qiagen).
7
Thermal Shift Analysis of HER3 Mutants
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CETSA was performed with COS7 cells transfected with HER3wt-RFP, HER3T787M-RFP or HER3KGG-RFP plasmids as described in (Jafari et al., 2014 (link); Reinhard et al., 2015 (link)). Briefly, COS7 were treated with DMSO or 50 nM bosutinib for 1 hr at 37°C. Cells were washed with PBS, detached and washed again twice with cold PBS. Cell pellets were resuspended in cold PBS with protease inhibitors (Roche) and 100 μl of each cell suspension was transferred into 0.2 ml PCR tubes. PCR tubes were heated for 3 min at 42°C or 50°C in a thermal cycler (DNA Engine DYAD, MJ research, Peltier thermal cycler) and incubated at room temperature for 3 min. Tubes were then immediately transferred onto ice, 35 μl of cold PBS 1.4% NP-40 with protease inhibitors were added and tubes were snap-frozen. Samples were then subjected to two freeze-thaw (at 25°C) cycles and cell lysates were centrifuged at 20,000 g for 1 hr at 4°C. Supernatants were carefully removed and analysed by western blot.
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