PCR products were evaluated by amplicon melting temperature (T m ), which was obtained from the melting cur ves (Supplementar y Material) provided by the LightC ycler 1.5 computer automatically after thermocycling. Furthermore, all PCR products were analyzed by agarose gel electrophoresis: 7-mL aliquots of the PCR mixtures were applied in loading buffer (0.25% xylene cyanol, 0.25% bromophenol blue, 30% glycerol) to 2% agarose gels (Invitrogen) in 1× TBE buffer (Invitrogen) and electrophoresed in a subcell GT electrophoresis chamber (Bio-Rad, Hercules, CA) at 85 V for 60 min. The gels were stained in aqueous ethidium bromide (0.2 mg/mL), rinsed with water, and photographed on a transilluminator (320 nm) (Model LMS-20E; Ultra-Violet Products Ltd., Cambridge, UK). For size standards, a 100-bp or 50-bp DNA marker ladder
Agarose gel
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Spain, Lithuania
Agarose gel is a commonly used laboratory equipment for the separation and analysis of DNA, RNA, and proteins. It consists of a porous matrix created by agarose, a polysaccharide extracted from seaweed. The gel acts as a sieve, allowing smaller molecules to migrate faster through the pores than larger ones when an electric current is applied. This separation technique is a core function of agarose gel in various bioanalytical and molecular biology applications.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Ethidium bromide, by Merck Group (8 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Qiaamp dna mini kit, by Qiagen (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Taq polymerase, by Thermo Fisher Scientific (3 mentions)
100 bp dna ladder, by Thermo Fisher Scientific (3 mentions)
Uv transilluminator, by Bio-Rad (3 mentions)
Tri reagent, by Molecular Research Center (3 mentions)
Gelred, by Biotium (3 mentions)
Qiamp dna mini kit, by Qiagen (2 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Ethidium bromide, by Thermo Fisher Scientific (2 mentions)
Formaldehyde, by Merck Group (2 mentions)
Gelred nucleic acid gel stain, by Biotium (2 mentions)
Digidoc it, by Analytik Jena (2 mentions)
Sybr safe, by Thermo Fisher Scientific (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Gel doc xr, by Bio-Rad (2 mentions)
152 protocols using agarose gel
1
PCR Product Evaluation via Melting Temperature and Gel Electrophoresis
2
Genetic Variation Detection by PCR-RFLP
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Amplifications were performed under standard conditions for polymerase chain reaction (PCR) (Master Mix, Promega Corp., Madison, WI, USA), following the instructions of the manufacturer, using a Peltier Thermal Cycler MJ96G (Biocycle Co. Ltd., Hangzhou, China) with the conditions previously standardized for the primer of interest, according to the specific literature. The PCR-amplified products were analyzed by electrophoresis on 2% agarose gel (Gibco BRL, Paisley, UK) for 45 minutes at ∼ 100 V. Subsequently, they were submitted to the restriction fragment length polymorphism (RFLP) technique, which detects a genetic variation with the use of a restriction enzyme. The products obtained by digestion with restriction enzymes were analyzed by electrophoresis on 3% agarose gel (Gibco BRL, Paisley, UK) for 45 minutes at ∼ 100 V. To analyze the results, we used the E-BOX 300 UV capture system (Vilber Lourmat, Collégien, France).
3
Cpf1 Nonspecific Cleavage Activation
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Nonspecific cleavage activity of Cpf1 was activated by incubating Cpf1, crRNA, and DNA activator with a concentration of 100 nM:100 nM: 2 nM in 1× NEBuffer 2.1 buffer at 37 °C for 30 min. M13mp18 was then added to the 30 μl reaction mixture and incubated for an additional 45 min. A fraction of the reaction was taken out every 5 min, quenched in 6× purple gel loading dye (New England Biolabs Inc.), and subsequently analyzed in 1% agarose gel (Fisher Scientific)1 (link).
4
Aptamer Serum Stability and Biodistribution
5
Genotyping transgenic mouse lines
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Mouse ear was punched using a 2-mm-diameter ear punch device from the ear lobe at 3-week old of age. All samples were stored at −20 °C until analysis. Frozen ear tissues were extracted in 50 mM NaOH (50 µL) at 98–100 oC for 1 hour. Then 50 µL of Tris buffer, pH8.0, was added to adjust the pH. The samples were centrifuged for 12,000 rpm × 5 mins at room temperature (25 oC). Isolated total DNA was transferred to a new tube and 2 µL of each sample preparations was used for PCR detection of the transgenic genes. For MMTV-scIgG gene, we used the forward primer 5′ CCTGTGGTGGTTGGAAGCTGG-3′ and the reverse primer 5′ CCACCC CTGGTTAGCCACATA-3′; for MMTV-PyVT gene amplification, we used forward primer 5′ GGAAGCAAGTACTTCACAAGGG-3′ and the reverse primer 5′ GGAAAGTCA CTAGGAGCAGGG-3′ based on the instruction from Jackson Lab. The PCR reaction was carried out using amfiSure PCR Master Mix (GenDEPOT) in 12.5 μL using a program of 31 cycles of amplification with annealing temperature at 56 °C for 30 sec, and extension at 72 °C for 30 sec, with final extension at 72 °C for 5 min using a C1000 Touch instrument (Bio-Rad, Hercules, CA). PCR products were subjected to DNA electrophoresis on a 1.8% agarose gel (Fisher). Samples with detectable DNA band at the expected size are defined as positive genotype.
6
Bacterial DNA Amplification by PCR
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PCR amplification was performed in a thermocycler (PTC-100, MJ Research, Waltham, MA, USA). The amplification was in a total volume of a 25 μl containing 12.5 μl of RedTaq PCR Master Mix (3 mM MgCl 2 , 0.4 mM of each dNTP, 0.06 U/μl of Taq DNA polymerase), (Sigma-Aldrich, California, USA), 1 μl of each forward and reverse primer (10 μM), (Microsynth, Balgach, Switzerland), 3 μl of whole-cell bacterial lysate, and 7.5 μl nuclease-free water. The PCR products were separated by gel electrophoresis (DYCP-31DN, Beijing Liuyi instrument, Beijing, China), with 1.6% agarose gel (Fisher Bio Reagents, USA), then viewed under an ultraviolet (UV) transilluminator (LMS-20E UVP, Upland, California, USA). Molecular size marker (100 bp) (Direct load, Sigma Aldrich, California, USA).
7
Genomic DNA Extraction and Sequencing of Diatraea saccharalis
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DNA isolation and sequencing D. saccharalis moths used for the DNA extraction were from an inbred colony which originated in Houma, Louisiana, and was reared on an artificial diet (Southland Products, Lake Village Arkansas). The insects were raised for about 70 generations, with new insects occasionally added to the colony. The adult moths were placed in 100% ethanol and shipped to the lab for DNA extraction. Genomic DNA was isolated from thorax and leg tissues derived from a single adult male specimen. The tissues were ground up to become a fine powder in a 10 mM Tris-HCl (Sigma-Aldrich, USA), 400 mM NaCl (Fisher Scientific, USA) and 2 mM EDTA (Fisher Scientific, USA) solution with the addition of RNase A (New England BioLabs, USA), and the high molecular weight DNA was precipitated using 120 µl 5M NaCl (Fisher Scientific, USA) (Miller et al., 1988) . Prior to sequencing, we assessed the DNA concentration (620 ng) using a NanoDrop ND-1000 spectrometer (v3.8.1, Thermo Scientific, USA) and fragment size (18.2 kb) in a 0.7% agarose gel (Fisher Scientific, USA). A PCR-free shotgun strategy was used to prepare the genomic library with insert sizes ranging from 200 to 500 bp. Raw sequence data from paired-end libraries with read lengths of 2x250 were generated by an Illumina HiSeq 2500 sequencer at the University of Illinois at Urbana-Champaign.
8
H9N2 Virus Plaque Assay in CEFs
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CEFs from two G2 hemizygous transgenic progeny (D-280 and E-185) and non-transgenic embryos were prepared and cultured in M199 (Invitrogen, Germany) and Ham’s F-10 (Sigma, St Louis, MO) mixed media containing 10% FBS (Hyclone, Logan, UT, USA), 0.65% sodium bicarbonate, and 1% penicillin/streptomycin. The CEFs were prepared 24 h prior to H9N2 infection at 107.5 EID50/200 µl, and the infections were performed in triplicate. After 48 h, confluent MDCK monolayers that had been propagated in 6-well plates were infected with an undiluted supernatant and a 10-fold diluted supernatant from the H9N2 virus-infected CEFs. After infection, the cells were washed with PBS and overlaid with a 0.7% agarose gel (Invitrogen) containing 1 /ml tosylsulfonyl-phenylalanyl-chloromethyl ketone (TPCK)-treated trypsin (Sigma). After 48 h, the cells were fixed in 10% PBS-buffered formalin and plaques were visualized using crystal violet staining.
9
Shift Mobility Assay for DNA Binding
10
Quantitative PCR Protocol for Gene Expression
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PCR fragments of all applied genes were generated by PCR on an Eppendorf Thermocycler gradient (Eppendorf, Hamburg, Germany). PCR cycling parameters were as follows: thermal activation for 10 min at 95°C and 50 cycles of PCR (melting for 45 s at 94°C, annealing for 45 s at 55–65°C, and extension for 60 s at 72°C). Applied primers are listed in Tables s 2A ,B . To verify the specificity of the PCR reaction, PCR products were electrophoresed alongside the 50 bp DNA Molecular Weight Marker XIII (Roche Diagnostics, Mannheim, Germany) through a 2% (w/v) agarose gel (Invitrogen). The gels were stained with SYBR green (Roche), and images were captured using a Kodak EDAS 120 Image System (Eastman Kodak Sàrl, Genève, Switzerland). The PCR products were purified with QIA quick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions, and the DNA concentration was determined using NanoVue. The copy number was calculated and serial 10-fold dilutions were made in the range of 1 × 107–1 × 101 copies.
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