Genomic DNA from leaf samples was isolated by following the standard protocol as per the procedure described by Murray and Thompson (1986) , with few modifications. Final concentration of 30 ng/µl of genomic DNA was used for PCR Genetic Diversity Analysis in Extra Early Pigeonpea [Cajanus cajan (L.)] Genotypes using SSR Markers (Eppendorf) amplification. PCR was performed using 1 U of Taq DNA polymerase (Fermentas, Lithuania) and 1x PCR buffer (Genei, India) in 10-µl reaction volume with a thermal profile of 94C for 5 min (initial denaturation), followed by 35 cycles of denaturation at 94C for 1 minute, annealing temperature (Table 2) for 1 min, extension at 72C for 2 min and a final extension of 7 min at 72C. The amplified products were electrophoretically resolved on 4% Seakem LE® Agarose (Lonza, USA), containing 0.5 mg/ml of ethidium bromide in 0.5x TBE buffer and visualized under UV.
Seakem le agarose
Manufactured by Lonza
Sourced in United States, Switzerland, Belgium
SeaKem LE Agarose is a laboratory-grade agarose product used for electrophoresis applications. It is designed to provide high gel strength and resolution for the separation and analysis of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Gelred, by Biotium (4 mentions)
Emerald gt pcr master mix, by Takara Bio (4 mentions)
Sybr gold, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Highpure pcr product puri cation kit, by Roche (3 mentions)
Bx61 microscope, by Olympus (3 mentions)
Bromophenol blue, by Merck Group (2 mentions)
Iproof dna polymerase, by Bio-Rad (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Lipofundin mct lct 20, by B. Braun (2 mentions)
Ssofast evagreen supermix, by Bio-Rad (2 mentions)
Cfx96 real time pcr system, by Bio-Rad (2 mentions)
Sybr green, by Lonza (2 mentions)
Imagequant tl version 8, by GE Healthcare (2 mentions)
Gentamycin, by Thermo Fisher Scientific (2 mentions)
Axioplan epifluorescence microscope, by Zeiss (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Sub cell gt cuvette, by Bio-Rad (2 mentions)
Uplanfln 100, by Olympus (2 mentions)
103 protocols using seakem le agarose
1
Genomic DNA Extraction and Genetic Diversity Analysis in Pigeonpea
2
Flagellin Gene Locus Analysis of Campylobacter jejuni
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The flagellin gene locus of C. jejuni contains 2 flagellin genes (flaA and flaB), which are arranged in tandem, and separated by approximately 170 nucleotides. The flaA gene is highly conserved and possesses variable regions, making this locus suitable for restriction fragment length polymorphism (RFLP ) (Wassenaar and Newell, 2000 (link)). The polymerase chain reaction was performed to amplify 1,713 bp flaA gene, according to Wassenaar and Newell (2000) (link). The cycling conditions submitted in thermocycler VWR Dopio (VRW, Belgium) were as follows: initial denaturation step at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 s, annealing 50°C for 1 min, extension at 72°C for 1 min; and final elongation step at 72°C for 5 min. For positive control, DNA extracted from C. jejuni NCTC11168 and C. coli CRL/SVA was used. PCR products were submitted to agarose gel electrophoresis. Gel Red (nucleic acid staining solution, Biotium, Fremont, EUA) and Bromophenol blue (Merck, Germany) loading dye were homogenized with PCR products. PCR products (1,713 bp) were run in a 1.5% agarose gel (SeaKem LE Agarose, Lonza), with TBE (Tris-borate-EDTA) 1X buffer, for 45 min under 100V. The DNA molecular weight marker used ranged from 200 to 10,000 bp (NZYTech ladder III). PCR products were visualized and photographed under UV light with ChemiDoc XRS+MP Imaging System (Biorad Laboratories).
3
Anchorage-Independent Growth Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine anchorage-independent growth, trypsinized spheres (2 × 104 cells/ml) were suspended in 0.4% SeaKem LE Agarose (Lonza, 50004) with serum-free DMEM/F12 containing 10 ng/ml EGF, 10 ng/ml FGF and N-2 supplement. This suspension formed the upper layer, which was poured onto a lower layer formed of 0.8% agarose in a composition otherwise identical to the upper layer. The layers with the cell suspension were poured into 6-well plates. The cells were incubated at 37 °C in a 5% CO2 atmosphere. After 14–30 days, the cells were stained with 0.05% crystal violet, and the colonies were photographed and counted in three randomly chosen fields. All cultures were performed in triplicate and represented by three independent experiments.
4
Electrophoretic Mobility of DNAJB6b Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Non-labelled DNAJB6b at various concentrations was mixed with IRdye680-labelled DNAJB6b at constant concentration, so that all samples contained 5 nM labelled protein and the total protein concentration varied from 5 nM to 32 μM, in steps of about a factor of 2. The agarose gel was prepared from 1% w/v SEAkem LeAgarose (Lonza Bioscience, Basel, Switzerland) in 50 mM Hepes/NaOH pH 8.5 and cast on the hydrophilic side of a 110 x 205 mm Gelbond film (Lonza Bioscience, Basel, CH). 10 μL sample was loaded per lane and the gel was operated horizontally on a water-cooled bed at 260 V for 25 min (Johansson, 1972 ) The gel was blotted onto a PVDF membrane using a 5 kg weight as a press. The membrane was dried and scanned using an IR fluorescence scanner Odyssey CLx, Li-Cor (Bad Homburg, Germany). The electrophoretic mobility was measured as the length travelled during the electrophoresis, in arbitrary length units (l. u.).
Using the image analysing software, ImageJ, the IR-intensities for each concentration were analysed along the direction of electrophoretic movement.
Using the image analysing software, ImageJ, the IR-intensities for each concentration were analysed along the direction of electrophoretic movement.
5
Quantifying Viral Titers via Plaque Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Madin-Darby Canine Kidney (MDCK) cells were seeded at a cell density of 0.7 × 10 6 cells per well in a six-well tissue culture plate (Nunc, Denmark) and grown at 37 °C in a CO 2 incubator. After the cells attained confluency, the confluent monolayers were washed with DMEM medium without FBS and infected with serial virus dilutions (10 -1 till 10 -5 ) and incubated for 1 h at 37 °C. After the incubation period, the cells were overlaid with overlay media containing 0.18% SeaKem LE Agarose (Lonza, Switzerland) and allowed to solidify. After 48 h of incubation at 37 °C, the plaques were stained with 0.1% crystal violet and counted. The virus titer was calculated as plaque-forming unit per milliliter (PFU/ ml).
6
Influenza Virus Plaque Assay in MDCK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDCK cells were seeded on a 6-well plate at a density of 1.2 × 106 per well 1 day prior to infection. Before the plaque assay, cells were washed with PBS. Influenza virus stocks or culture supernatant from the in vitro infection experiment were diluted 10-fold serially with MEM with 1% PS. 1 mL of the diluted virus/ sample was added to each of the six wells, and the plate was rocked gently every 15 min for 1 h in a 37 °C incubator with 5% CO2. After the virus adsorption, the inoculum was discarded and the cells were washed with PBS. In total, 2 mL agarose overlay reconstituted from MEM containing 1 µg/mL TPCK-treated trypsin in 0.8% SeaKem® LE Agarose (50002, Lonza, Basel, Switzerland) was added to each well. Upon the solidification of the agarose, the assay plates were incubated upside-down in a 37 °C incubator with 5% CO2 for 3 days. On day three post inoculation, 10% PBS buffered formalin was added onto the agarose for cell fixation. The agarose was removed from the well and the MDCK cell was subsequently stained by 1% crystal violet (1 g crystal violet dissolved in 10 mL 95% ethanol and 90 mL MilliQ water) for 1 h. The stained monolayer was then washed and air-dried.
7
EGFR Gene Mutation Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In a 20 μl assay, 1X Emerald GT PCR master mix (Takara/Clontech, USA) was added, along with m13-tagged forward and reverse target primers (5 μM). Approximately 50 ng of template DNA is added, in a typical assay, and made up with distilled water. Primers for exons 18, 19, 20, and 21 of the EGFR gene (NCBI Genbank Accession ID: NM_005228.3) were synthesized (Merck-Sigma, Bangalore, India). Design and characterization of the primer sequences for both sequencing and HRM were obtained from a previously published literature [18 (link)]. Thermal cycler settings included an initial denaturation of 95 °C for 15 min, followed by 45 cycles of denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, extension at 72 °C for 45 s and a final extension at 72 °C for 10 min. The amplicons were assessed using 2% Agarose gel (SeaKem® LE Agarose, Lonza, USA). The PCR products were then subjected to post-PCR clean up to remove residual primers and other enzyme proteins using HighPure® PCR product purification kit (Roche Molecular Diagnostics, Switzerland).
8
Bracing-Induced Scoliosis in Rats
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Female Wistar rats aged 21 days were procured from Jeeva Life Sciences Pvt. Ltd. (Hyderabad, India). The bracing material (i.e., leather) and LED light joint wires were purchased from local markets. Rat spines were braced in a scoliotic position. Real-time polymerase chain reaction (RT-PCR) primers for rat were designed using Primer 3 software for the targeted genes and procured from Eurofins Genomics India Pvt. Ltd. SYBR Premix Ex Taq, TaKaRa BioMasher Standard Verso cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA; cat. no. AB-1453/A), Seakem LE agarose (Lonza, Basel, Switzerland; cat. no. 50004), RNA Sure mini kit (Nucleopore, Genetix, India; cat. no. NP-84105), and Insta Q96 RT-PCR Machine (HiMedia Pvt. Ltd., Mumbai, India) were used for gene-expression studies.
9
Molecular Cloning Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA ladders and iProof DNA polymerase were obtained from Bio-Rad, Hercules, CA, USA. PrimeStar DNA polymerase mixture was purchased from Takara, Kusatsu, Shiga, Japan. T4 DNA ligase and restriction endonucleases were purchased from New England Biolabs, Ipswich, MA, USA. SeaKem® LE Agarose was obtained from Lonza, Basel, Switzerland. Gel extraction/PCR cleanup kit was purchased from Biotools, New Taipei City, Taiwan. Plasmid mini-prep kit was obtained from Geneaid, New Taipei City, Taiwan. Oligonucleotides synthesis and DNA sequencing services were provided by Tri-I Biotech, New Taipei City, Taiwan.
10
SFTSV Focus-Forming Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vero E6 cells (3 × 105 per well) were seeded in 6-well plates and infected with 10-fold serial dilutions of virus for 1 h at 37°C, followed by the addition of 5 mL/well of overlay medium containing 2× DMEM (Welgene, Daegu, South Korea), 10% FBS, 1% penicillin/streptomycin, and 0.5% SeaKem LE Agarose (Lonza). Cells were incubated at 37°C for 7 days and then fixed with 10% formaldehyde in PBS. After washing with PBS, cells were permeabilized in 10% Triton X-100 in PBS for 10 min. Anti-SFTSV nucleoprotein (NP) monoclonal antibody (6B3, in-house) and HRP-conjugated anti-mouse IgG antibody were used for immunostaining the infected cells. Foci were detected using 3,3′-diaminobenzidine substrate (Vector Laboratories). Visible foci were counted and used to calculate viral titers.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!