LT-1 and ST-1 toxin genes in diarrheagenic pathogen E. coli strains were confirmed by PCR analysis. The Primers such as LT-1F-5′ACGTTCCGGAGGTCTTATGC-3′ and LTR-5′ AGCCGGTTTGTGTTCCTCTC-3′; ST-1F-5′CGTGAAACAACATGACGGGAG-3′ and ST1-R-5′ CAGTTGACCTGACTAAAAGAGGGGA-3′ were used for LT-1 and ST-1 toxin genes respectively were designed using nucleotide sequence obtained from NCBI-Gene bank (Accession number- S60731.1) and used for PCR analysis. A PCR reaction mixture of 20 µL was prepared for each pathogen and analysis was carried out with Mycycler™ thermal cycler (Biorad, California, USA). The reaction mixture was prepared using 0.2 µL of HotstartTaq® DNA polymerase, 2 µL primer, 1 µL PCR buffer, and 2 µL of DNA sample. PCR reaction was run through activation step (95 °C/15 min), followed by denaturation step 35 cycles of (95 °C/15 min), annealing (55 °C/45 s), extension (68 °C/2 min), and final elongation (72 °C/5 min). PCR amplified products were subjected to electrophoresis using agarose gel (1.0% w/v). After the run, PCR product size and bands were examined under UV tans-illuminator (Foto/UV-21, Fotodyne Inc., USA) and photographed using BioRad Gel Doc system (Bio-rad, Hercues, CA, USA) in correspondence with DNA ladder (Bangalore Genei, Bangalore, India) of 100 bp.
Dna ladder
Manufactured by Bio-Rad
Sourced in United States
A DNA ladder is a laboratory tool used to determine the size of DNA fragments in gel electrophoresis experiments. It consists of a mixture of DNA fragments of known sizes, which can be used as a reference to estimate the size of unknown DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc system, by Bio-Rad (1 mentions)
Mycycler thermal cycler, by Bio-Rad (1 mentions)
Thermal cycler, by Bio-Rad (1 mentions)
Microtome, by Thermo Fisher Scientific (1 mentions)
Digital camera, by Bio-Rad (1 mentions)
Sybr safe stain, by Applied Biological Materials (1 mentions)
Gelred, by Biotium (1 mentions)
Iproof dna polymerase, by Bio-Rad (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Restriction endonuclease, by New England Biolabs (1 mentions)
Seakem le agarose, by Lonza (1 mentions)
Quikchange approach, by Agilent Technologies (1 mentions)
7 protocols using dna ladder
1
Detecting Toxin Genes in E. coli
2
Extraction and Amplification of H. pylori DNA
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1 ml of each milk sample was used for extraction of cDNA by a DNA isolation kit (Cat. No. ABIN412492, Roche, Germany) according to the manufacturer’s instruction, with slight modification according to Quaglia et al. [18 (link)]. Extracted DNA was amplified according to Rahimi and Kheirabadi [20 (link)] for the glmM gene of 294 bp using 5-GAATAAGCTTTTAGGGGTGTTAGGGG-3 as primer forward and 5-GCTTACTTTCTAACACTAACGCGC-3 as primer reverse. PCR reactions were performed in a final volume of 50 μl containing 25 μl Green Master Mix (Sigma), 10 μl genomic DNA as a template, 13 μl free ionized water, and 1 μl of each primer. PCR was performed using a thermal cycler (Bio-Rad, France) under the following conditions: Initial denaturation for 10 min at 94°C, 35 cycles for 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, and a final extension at 72°C for 10 min.
The PCR products were electrophoresed through a 1.5% agarose gels (Bio-Rad, France) containing ethidium bromide.
A DNA ladder (Bio-Rad, France) used to detect the molecular weight of observed bands under a UV lamp. Samples inoculated with H. pylori were used as positive controls, and sterile distilled water was used as negative control.
The PCR products were electrophoresed through a 1.5% agarose gels (Bio-Rad, France) containing ethidium bromide.
A DNA ladder (Bio-Rad, France) used to detect the molecular weight of observed bands under a UV lamp. Samples inoculated with H. pylori were used as positive controls, and sterile distilled water was used as negative control.
3
Decellularized Bone Matrix Characterization
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DBM was verified by histological analysis and DNA quantification. For histological analysis, native bone matrix (NBM) and DBM were fixed with 4% paraformaldehyde (PFA) and dehydrated prior to embedding in paraffin. NBM and DBM in the paraffin-embedded blocks were sectioned at 5 μm thickness by microtome (Thermo-Scientific, USA). The sections of NBM and DBM were stained with hematoxylin and eosin (H & E) to confirm the removal of cellular contents after decellularization.
For DNA quantification, the total DNA of NBM and DBM were extracted and quantified it using a DNA Quantification Kit (GeneAll, 104–101, Korea). The extracted DNA from NBM/DBM was run in 1% agarose gel with a DNA ladder (Biorad, US).
For DNA quantification, the total DNA of NBM and DBM were extracted and quantified it using a DNA Quantification Kit (GeneAll, 104–101, Korea). The extracted DNA from NBM/DBM was run in 1% agarose gel with a DNA ladder (Biorad, US).
4
PCR Assay for Veterinary Blood Samples
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PCR assay was conducted at the Laboratories of Internal and Preventive Veterinary Medicine Department, College of Veterinary Medicine, University of Baghdad, and PCR assay was done on 310 frozen blood samples by the following methods.
The total master mix reaction of PCR was 25 μL, according to instructions of (Promega, company, USA), total reaction containing 12.5 μL of the master mix, 0.5 μL of 10 pmol of each forward and reverse primers, 3 μL of DNA template, and 8.5 μL of nuclease-free water. All reagents were procured by Promega, USA. The thermocycling protocol (Table 2) was according to Davitkov et al. (14) for PIRO-A and PIRO-B primers and according to Jain et al. (15) for BAGI F and BAGI R primers. The DNA products were loaded on 1.2% agarose gel with SYBR Safe stain (ABM, Canada), the positive band was compared with 100 bp commercial marker DNA ladder (Bio-Rad Laboratories, USA). Controls as non-template negative controls were included. Gels were photographed under UV light using a digital camera (Bio-Rad Laboratories, USA).
The total master mix reaction of PCR was 25 μL, according to instructions of (Promega, company, USA), total reaction containing 12.5 μL of the master mix, 0.5 μL of 10 pmol of each forward and reverse primers, 3 μL of DNA template, and 8.5 μL of nuclease-free water. All reagents were procured by Promega, USA. The thermocycling protocol (Table 2) was according to Davitkov et al. (14) for PIRO-A and PIRO-B primers and according to Jain et al. (15) for BAGI F and BAGI R primers. The DNA products were loaded on 1.2% agarose gel with SYBR Safe stain (ABM, Canada), the positive band was compared with 100 bp commercial marker DNA ladder (Bio-Rad Laboratories, USA). Controls as non-template negative controls were included. Gels were photographed under UV light using a digital camera (Bio-Rad Laboratories, USA).
5
DNA Extraction and Size Determination
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Chromatin/gDNA fragments were processed
by IPure kit to extract
DNA. Purified DNA was dissolved in 10 μL of water and analyzed
on GelRed (41003, Biotium) stained 1% agarose gel.23 (link)−25 (link) The size of
the bands was determined using 100–3000 bp DNA ladders (170–8206,
Bio-Rad).
by IPure kit to extract
DNA. Purified DNA was dissolved in 10 μL of water and analyzed
on GelRed (41003, Biotium) stained 1% agarose gel.23 (link)−25 (link) The size of
the bands was determined using 100–3000 bp DNA ladders (170–8206,
Bio-Rad).
6
Molecular Cloning Protocol
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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.
7
Standardized Molecular Biology Protocols
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Standard chemicals and biochemicals were obtained from Sigma and Merck. Molecular-size markers and DNA ladders were obtained from Bio-Rad and Biolab, respectively. Site-directed mutagenesis was performed using the QuikChange approach (Agilent).
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