sgRNA PCR amplicons were mixed with 6 × loading dye (Roti-Load, Carl Roth) and loaded on a 1% agarose gel (w/v, Sigma Aldrich) in 1 × TAE buffer (Carl Roth) next to a 1 kb Plus DNA ladder (Thermo Scientific). The samples were run for 25 min at 140 V, stained using ethidium bromide (AppliChem GmbH), and visualized in a Bio-Rad Gel Doc XR + .
Plus dna ladder
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Plus DNA Ladder is a molecular weight standard used for the size determination of DNA fragments in gel electrophoresis. It contains fragments of known sizes that can be used as a reference to estimate the size of unknown DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Ethidium bromide, by Merck Group (2 mentions)
Ethidium bromide, by AppliChem (1 mentions)
Roti load, by Carl Roth (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Recombinant taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Ec68755box, by Thermo Fisher Scientific (1 mentions)
Bst dna polymerase, by New England Biolabs (1 mentions)
Purple gel loading dye, by New England Biolabs (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Polymerase, by Merck Group (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
Gel doc ez imager, by Bio-Rad (1 mentions)
Generuler, by Thermo Fisher Scientific (1 mentions)
Agarose gel, by Merck Group (1 mentions)
8 protocols using plus dna ladder
1
Agarose Gel Electrophoresis of sgRNA PCR Amplicons
2
Reverse Transcription and PCR for IBV Detection
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Reverse transcription was carried out with 5 µL supernatant-derived RNA using SuperScript IV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The detection of the IBV 3′ untranslated region (UTR) was achieved using universal primers BG56 (5′-CAACAGCGCCCAAAGAAG-3′) and BG93/100 (3′-GCTCTAACTCTATACTAGCCT-5′). PCR across the S gene was carried out using primer sets covering the S1 and S2 subunits. Primers covering S1 are termed BG42 (5′-AATAATGGCAATGATGAC-3′) and BG136 (3′-AACTGCCACAAACATACTGC-5′). Those covering S2 are termed BG46 (5′-CATCAAAATCACTAATGG-3′) and BG142 (3′-AGGGATCAAATACTTCTGTG-5′). PCR products were generated using Taq DNA polymerase recombinant (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. An extension time of 1 min per kb was employed. PCR products were analysed by gel electrophoresis and product sizes (bp) were compared to 1 kb Plus DNA Ladder (ThermoFisher, Waltham, MA, USA). PCR products were sent to Eurofins GATC for Sanger sequencing with each primer, according to the company’s requirements. Sequencing reads were analysed using Staden software.
3
Primer Exchange Reaction Evaluation
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100 μl Primer Exchange Reactions were prepared for both Tigerfish probes and PaintSHOP bridge sequences with a final concentration of 1x PBS, 10 mM MgSO4, 400–1000 U/ml Bst DNA Polymerase (large fragment), 120,000 units/ml (NEB M0275M), 100 nM Clean G hairpin, 50 nM–1 μM hairpin and water to 90 μl. After incubation for 15 min at 37 °C, 10 μM oligo probe(s) were added and the reaction was incubated for another 2 h with another 20 min at 80 °C to heat-inactivate the polymerase. PER extension solutions were directly diluted into FISH solutions. Lengths of the concatemers were evaluated by diluting 6.7 μl of the in vitro reaction with 3.3 μl 6X TriTrack. Samples were then run on a 10% TBE-Urea denaturation gel (ThermoFisher EC68755BOX) for 10 min alongside 1 kb Plus DNA Ladder to estimate length and imaged with SYBR Gold channel and then imaged after a 15 min incubation.
4
Agarose Gel Electrophoresis of LASSO Captures
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A 4.0% agarose gel was cast and pre-stained with ethidium bromide. The whole post-capture PCR reaction was mixed with 5 μl of 6X Purple Gel Loading Dye (NEB). Novex 4–12% 12-well TBE Polyacrylamide Gels (Thermo Fisher) were used to resolve 2.5 μl of PCR amplified LASSO captures in 1X Purple Gel Loading Dye. For capture-to-noise ratio quantitation, four two-fold serial dilutions were made starting with a 1X PCR solution containing 8 μl of PCR product (60o C Ta), 12 μl of ddH2O, and 4 μl Purple Loading Dye. Four two-fold serial dilutions of 1 kb Plus DNA Ladder (Thermo Fisher) were included for relative capture DNA mass estimation. PAGE for DNA quantitation was performed in duplicates. All PAGE gels were stained with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher) for 10 min before visualization via a gel imager (FastGene).
5
PCR Amplification and Gel Visualization
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The PCR was carried out in a 25 μl reaction mixture containing the following: 1 μl of DNA (50 ng μl−1), 12·5 μl PCR buffer (50 mmol l−1 KCl, 1·5 mmol l−1 MgCl2, 10 mmol l−1 Tris-HCl, pH 8·8, 0·1% TritonX-100), 1U polymerase (Sigma-Aldrich), l0 mmol l−1 dNTP (Invitrogen, Carlsbad, CA), 0·5 μl 100 mmol l−1 of each primer and 11·5 μl H2O. Amplifications were performed in C1000 Touch™ Thermal Cycler (Bio-Rad) under the following conditions: initial denaturation 5 min at 94°C, 35 cycles of 45 s at 94°C, 45 s at 53–56°C (Table1 ), 1 min at 72°C and for the final extension 10 min at 72°C. Amplification products were separated on a 1·5% agarose gel (Invitrogen) in 1 × TBE buffer (0·178 mol l−1 Tris-borate, 0·178 mol l−1 boric acid, 0·004 mmol l−1 EDTA) and stained with ethidium bromide. The 10 μl PCR products were combined with 2 μl of loading buffer (0·25% bromophenol blue, 30% glycerol). A 100-bp DNA LadderPlus (Fermentas, St. Leon-Rot, Germany) was used as a size standard. PCR products were electrophoresed at 3 Vcm−1 for about 2 h, visualized under UV light and photographed (Gel DOC EZ Imager; Bio-Rad).
6
Agarose Gel Electrophoresis of PCR Products
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Aliquots of 15 ml of the PCR product were analyzed on 2% agarose gel (agarose from HiMedia) containing ethidium bromide (Merck KGaA, Darmstadt, Germany). Products were visualized under ultraviolet light. The size of the amplified product was determined by comparison with a base-pair ladder size marker (Gene Ruler, 100 bp, 50 bp DNA Ladder Plus, Fermentas, India).
7
Multiplex PCR for R. equi Detection
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Multiplex PCR reaction was used to confirm isolated strains belonging to R. equi species and to detect vapA gene characteristic for virulent R. equi strains. The target DNA for PCR amplification were species specific fragment of chromosomal gene coding the 16S subunit of rRNA and vapA gene of the virulence plasmid. The details concerning oligonucleotide primers used in the study are presented in Table 3 . PCR amplification was performed with 5 μl of DNA preparation in a 50 μl reaction mixture as it was described earlier [26 –27 (link)]. For each PCR reaction, the amplification of positive control sample (DNA of reference R. equi strain ATCC 33701) and negative control (with 5 μl of water instead of the DNA matrix) were performed in parallel under the same conditions. The PCR products were analysed electrophoretically in 1.5% agarose gel (Sigma) in TBE buffer in the presence of the molecular weight marker (100 bp DNA Ladder Plus, Fermentas, Lithuania). DNA fragments were visualized by UV fluorescence (Transilluminator Cole-Parmer, France) after staining with ethidium bromide (Sigma, USA).
8
Agarose Gel Electrophoresis of PCR Products
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PCR products and a molecular weight marker (100 bp DNA Ladder Plus, Fermentas, Glen Burnie, Maryland, USA) were loaded into a 1.5 % agarose gel and separated by horizontal electrophoresis in 1X TAE buffer at 100 V and 3 W for one hour. Gels were stained in 0.5 μg/mL ethidium bromide solution under gentle agitation for 30 min. Gels were exposed to 312 nm UV light in a transilluminator for visualization of the separate fragments of the PCR product and size comparison with the molecular weight marker. Gels were photographed using the imaging system G:BOX Chemi XR5 (GBCXR50313, SYNGENE©, Frederick, MD, USA).
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