Molecular imager gel doctm xr system

Targeted DSB formation in transduced cells was assessed by using genotyping assays based on the mismatch sensing T7EI enzyme. To this end, genomic DNA from mock-transduced and AdVP-transduced cells was isolated by using the DNeasy Blood & Tissue Kit (QIAGEN, cat. no. 69506) following the manufacturer’s recommendations. The DMD-specific PCR amplifications were performed with the Phusion High-Fidelity DNA Polymerase system (Thermo Fisher Scientific, cat. no. F-530). The primers, PCR mixtures, and cycling parameters used are specified in Tables S11 and S12. The resulting PCR amplicons were denaturated and reannealed by applying the thermocycling program indicated in Table S13. T7EI-based DNA cleaving assays were done as follows. First, 8 μL of each PCR mixture was incubated in 15-μL reactions consisting of 1× NEBuffer 2 (New England Biolabs, cat. no. B7002S) and 5 U of T7EI (New England Biolabs, cat. no. M0302). Next, after 15-min incubations at 37°C, the DNA samples were subjected to electrophoresis through 2% (w/v) agarose gels in 1× Tris-acetate-EDTA buffer. The resulting ethidium bromide-stained DNA species were then detected by using a Molecular Imager Gel-DocTM XR+ system (Bio-Rad) and analyzed via the Image Lab 6.0.1 software (Bio-Rad).

To confirm the identification of P. shigelloides, a thin layer of the bacteria was spread on nutrient agar plates and incubated at 36 °C for 24 h. Suspension was created by combining nuclease-free water, vortexed, and heated at 100 °C for 10 min before being boiled to eradicate bacteria. This was followed by a 5-min centrifugation at 12,000 rpm, and the supernatant was immediately transferred to a clean 2 mL Eppendorf tube and placed on ice for template DNA extraction. This is what we used to amplify our virulence genes. The primers we used can be found in Table 3. These primers were selected because previous research had shown that they were highly specific. The total volume of PCRs was 25 μL. Table 3 provides the PCR settings needed to find the virulence genes [4 ,12 (link),15 (link)]. Ethidium bromide staining (E8751- Sigma-Aldrich, USA) and 100 V were used in the following electrophoresis. A 1% (w/v) agarose gel was used in a 0.5 X TBE buffer for 45 min with a voltage of 100 V. The gene DNA ladder utilized a 100 bp molecular weight marker. An image of the gel was obtained after electrophoresis by means of the Molecular Imager Gel Doc^TM XR⁺ System and Image Lab^TM Software 170-8195 (BioRad, Hercules, CA, USA).

For genotyping the rag2 line and the tumor (graft) cells, WT DNA (i.e., DNA from non-injected frogs) was amplified in parallel with each unknown DNA sample via a standard PCR. Subsequently, equal quantities of both products—PCR-amplified WT and unknown sample DNA—were mixed and eventually subjected to HMA in parallel with all the unknown samples individually (unmixed). This was completed by incubation of the samples at 98 °C for 5 min, followed by a 4 °C holding temperature incubation using a transition with a ramp rate of 1 °C/s. Finally, the PCR amplicons were prepped with DNA loading dye and run on an 8% (bis)acrylamide/TBE gel. Visualization was performed on a Molecular Imager^® Gel DocTM XR+ System (Bio-Rad, Hercules, CA, USA) supported by the Image Lab software (Bio-Rad, Hercules, CA, USA). Original blots can be found online in the supplementary material file containing Figures S1 and S2.

DNA was extracted from mouse tails with 50 mM NaOH after incubation at 95 °C for 30 min. Wild-type and mutant allele were amplified independently using a polymerase (HOT FIREPol, Solis Biodyne, Tartu, Estonia), a common forward primer: mGpr179_EF (5′-CTGCCCCCACAGAATGTTCCCA-3′) and two specific reverse primers: mGpr179_Er2 (5′-CACCGCCTCTTTACTCTGCCCA-3′) for the wild-type allele and mGpr179_Kr (5′-GGGCAAGAACATAAAGTGACCCTCC-3′) for the mutant allele. The following PCR program was used: 15 min at 95 °C for denaturation, 35 cycles of 45 sec at 95 °C, 1 min at 60 °C, and 1.3 min at 72 °C. A final extension for 10 min at 72 °C was performed. This generates the following amplicons: PCR using mGpr179_EF and mGpr179_Er2 primers amplifies a product of 146 base pairs (bp) for the wild-type allele and no product for the mutant allele, PCR using mGpr179_EF and mGpr179_Kr primers amplifies no product for the wild-type allele and a 303 bp product for the mutant allele. PCR products were separated by electrophoresis on 2% agarose gels, stained with ethidium bromide, and visualized using a documentation system (Molecular Imager^® Gel Doc^TM XR+ System, Bio-Rad, Hercules, CA, USA).

The in vitro concerted integration assays were carried out as previously for described Rev-A IN following removal of the His₆-tag by site-specific proteolysis [66 (link)]. Briefly, 1 μM Rev-A IN mixed with 0.5 μM vDNA and 4 nM pGEM-3 tDNA, in 40 μL of 20 mM HEPES pH 7.4, 50 mM NaCl, 5 mM MgCl₂, 4 μM ZnCl₂, and 10 mM DTT, was incubated for 1 h at 37°C with varying concentrations of BRD4_462–720 or BRD4_{462–720/L630E} (0.05/0.15/0.35/0.5 μM). Reactions were stopped by adding 25 mM EDTA and 0.5% SDS, and deproteinized by digestion with proteinase K. Products were precipitated with ethanol and analyzed by electrophoresis through 1.5% agarose gels. DNA was detected by staining with ethidium bromide. Concerted integration products were measured by band intensity quantification using the Molecular Imager® Gel Doc TM XR+ System with Image Lab software (Bio-Rad).

ERIC-PCR was performed to assess genotypic diversity and to reveal clusters among isolates as well as to differentiate individual strains. ERIC-PCR employed genomic DNA extracted from cell pellets of each isolate as a DNA template, a pair of primers ERIC2 (5′ AAG TAA GTG ACT GGG GTG AGC G 3′) and ERIC1R (5′ ATG TAA GCT CCT GGG GAT TAC C 3′), and a thermal cycling condition consisting of a first denaturation step at 95 °C for 7 min, 30 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C for 1 min, and extension at 65 °C for 8 min, and a final extension step at 65 °C for 16 min [60 ]. Negative controls (no DNA template) were included in PCR reactions. The amplified fragments were separated by electrophoresis on 1%% agarose gels containing SafeView FireRed gel casting dye (Applied Biological Materials Inc., Richmond, BC, Canada) and visualized with a Molecular Imager^® Gel DocTM XR+ system (Bio-Rad Laboratories, Hercules, CA, USA). ERIC-PCR fingerprints and clusters were presented in the unweighted pair groups using mathematical averages (UPGMA) dendrogram created with the Phoretix ID Pro. software (TotalLab Ltd., Newcastle upon Tyne, UK). The isolates generating distinct ERIC-PCR fingerprints were characterized as individual strains and then were subjected to further steps of the study.

Cationic SNs were prepared upon addition of the cationic lipid, stearylamine (ST) to the organic phase at a ratio of VitE:SM:PEG:ST 10:1:0.1:1 (w/w). SNs-ST were fully characterized. miRNA was subsequently associated to SNs-ST by the establishment of electrostatic interactions performing a simple incubation. A total of 10 μL of miRNA (10 μg) were incubated with 90 μL of SNs-ST at different miRNA:ST mass ratios (1:10, 1:7.5, 1:5, 1:2.5, and 1:1) to obtain theoretical loadings ranging from 0.8% to 7.6% (w/w) with respect to the total mass of the nanosystems. The association of miRNA was analyzed by agarose gel electrophoresis (1% w/v in Tris-Acetate-EDTA (TAE) Buffer). Briefly, 0.5 μg of nucleic acids labeled with SYBR^® Gold, either in solution, associated to the nanoparticles, or after displacement with an excess of heparin (25-fold heparin with respect to the amount of miRNA for 2 h at 37 °C) were loaded into each well. Gel electrophoresis was run at 100 V, 40 min in a Sub-Cell GT cell 96/192 (Bio-Rad Laboratories Ltd., Deeside, England). Gel images were obtained with a Molecular Imager^® Gel DocTM XR System (UV light 302 nm; Bio-Rad, Madrid, Spain).