The strain SRP2 was identified by 16S rDNA sequencing, confirmed by its phenotypic and biochemical characteristics. The gene encoding 16S rRNA was amplified from the genomic DNA of SRP2 strain by PCR (polymerase chain reaction) using 16S rDNA universal bacterial primer pairs, which were HDA-1 (5´-GACTCCTACGGGAGGCAGCAGT) and E1115R (5´-AGGGTTGCGCTCGTTGCGGG), and a 726 bp fragment was obtained. The product obtained after PCR was purified using PCR purification Kit (Geneaid, Canada) following the manufacturer's protocol. The purified PCR sample was sent to Euroffins Genomics (USA) for sequencing. For identification of the strain SRP2, sequence analysis was performed using NCBI blast tool, compared with sequences of the Gene Bank database search (http://www.ncbi.nlm.nih.gov/genbank/ ). The phylogenetic relationship was analyzed using sequence alignment program ClustalX and TreeView software. The species identification was confirmed using phenotypic and biochemical features 16 .
Pcr purification kit
Manufactured by Geneaid
Sourced in Canada
The Geneaid PCR purification kit is a laboratory equipment designed to purify PCR amplicons. It removes unwanted components from the PCR reaction, such as primers, nucleotides, and enzymes, while retaining the desired DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
T100 thermocycler, by Bio-Rad (2 mentions)
Thermocycler, by Bio-Rad (2 mentions)
Sybrtm safe, by Thermo Fisher Scientific (2 mentions)
Onepcr tm master mix, by GeneDireX (2 mentions)
Staphylococcus aureus protein a, by Merck Group (1 mentions)
2xi taq master mix, by iNtRON Biotechnology (1 mentions)
Dreamtaq, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Dreamtaq buffer, by Thermo Fisher Scientific (1 mentions)
Chelex solution, by Bio-Rad (1 mentions)
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Pjet1.2 blunt vector, by Thermo Fisher Scientific (1 mentions)
Kapa 2g polymerase, by Roche (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Sybr safe, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
8 protocols using pcr purification kit
1
Identification of Bacterial Strain SRP2 via 16S rDNA
2
ChIP Assay for Chromatin Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The detailed procedure of the ChIP assay has been described in our previous papers [25 (link),28 (link),29 (link)]. Briefly, C2C12 cells were washed, fixed in formaldehyde (1%, w/v), and sonicated to shear chromatin. Specific antibody was added to the cleared lysate and the binding was allowed to proceed at 4°C overnight before Staphylococcus aureus Protein A (Sigma, #P7155) was added to capture the immune complex and then washed extensively. Then, the immune complex was eluted and the released DNA was extracted with phenol/chloroform twice and further purified using a PCR purification Kit (Geneaid). The primer sets used in the ChIP assay are listed in Supplementary Table S3.
3
Mitochondrial rRNA Genes Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The partial mitochondrial 12S and 16S rRNA genes were amplified in a T100™ thermocycler (Bio-Rad, California, USA) with the primers in Table 2 . Each 30 μl reaction contained 15 μl of 2X i-Taq™ master mix (iNtRON Biotechnology, Gyeonggi, South Korea), 0.1 μM of each primer, and 1 ng/μl of DNA. The thermocycling profiles were 94 °C for 2 min of initial denaturation; 35 cycles of 94 °C for 30s, 55 °C or 56 °C for 1 min, and 72 °C for 2 min; followed by a final extension step at 72 °C for 5 min. PCR amplicons were visualized on 1% agarose gel stained with GelRed® (Thomas Scientific, New Jersey, USA). Successful amplicons were purified with Geneaid PCR Purification Kit (Geneaid Biotech Ltd., Taipei, Taiwan) following the manufacturer’s recommendations. Purified DNA products were sequenced by a commercial company (Macrogen, Seoul, South Korea) on an automated Sanger sequencer using the primers for PCR amplification. The partial mitochondrial 12S and 16S rRNA gene sequences generated in this study were deposited in the NCBI database (12S rRNA gene sequences: MZ331635–MZ331683; 16S rRNA gene sequences: MZ331595–MZ331634, MZ345698–MZ345705).
4
Molecular Identification of Corynebacterium pseudotuberculosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A loopful of 48 h culture of C. pseudotuberculosis was suspended in 6% Chelex-solution (Bio-Rad Laboratories, Hercules, CA, USA) and then heated to 65°C for 30 min and to 100°C for 8 min; afterwards, it was centrifuged. The supernatant was collected in a new tube and stored at −20°C until use. Fragments of the selected housekeeping genes were amplified by PCR in an Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific, Carlsbad, CA, USA). The 25 μl amplification reaction mixture contained 1x Dream Taq Buffer, 200 nM dNTP-mix, 1 μM of each primer, 1 U Dream Taq (Thermo Fisher Scientific, Carlsbad, CA, USA), template DNA, and water. The PCR program included 3 min of initial denaturation at 95°C and then 30 cycles at 95°C for 30 s, at 60°C for 30, and at 72°C for 30 s and 7 min of the final elongation. PCR products were purified for direct sequencing using the Geneaid PCR purification Kit (Geneaid Biotech, New Taipei, Taiwan). Templates were sequenced with the PCR primers using the BigDye Terminator Ready Reaction Mix v3.1. Nucleotide sequences were run on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).
5
Mitochondrial 12S and 16S rRNA Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amplification of mitochondrial 12S and 16S rRNA genes was performed in a thermocycler (Bio-Rad, California, USA) using the primers in Table 1. PCR was performed in a final volume of 30 µl, containing 15 µl of 2× OnePCR TM master mix (GeneDireX. Inc., Taoyuan, Taiwan), 0.1 µM of each primer, and 5 ng/µl of DNA. The PCR thermocycling profiles were: 94 °C for 2 min of initial denaturation; then 35 cycles of 94 °C for 30 s, 45 °C to 55 °C for 1 min, and 72 °C for 2 min; followed by a final extension step at 72 °C for 5 min. The annealing temperatures used for each primer set are listed in Table 1. Amplicons were checked and visualized on 1% agarose gel and stained with SYBR TM safe (Thermo Fisher Scientific, Waltham, USA). Successful amplicons were purified using the Geneaid PCR purification kit (Geneaid Biotech Ltd.). Sequencing for each sample was performed by a commercial company (Macrogen, Seoul, South Korea) on an automated Sanger sequencer. Mitochondrial 12S and 16S rRNA gene sequences generated in this study were deposited in the NCBI database (NCBI: MT135051-135093 for 12S rRNA; MT151872-151910 for 16S rRNA).
6
Cassava EF1A Promoter Cloning
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene family identification was done by comparing the first exon of EF1A gene from cassava (AF041463) using blastn to the EF1A gene family available on Manihot esculenta genome database (Phytozome) [45] . A set of primers then was designed from that blastn result in order to clone the promoters from EF1A gene family. Genomic DNA was isolated from cassava leaves using CTAB method [46] . The promoter regions from each gene family were amplified using specific primers (Table 1 ). PCR amplification was performed using Kapa 2G Polymerase™ (Kapa Biosystem) in Veriti 96 well Thermal cycler (Applied Biosystems).
Each of PCR products from single PCR reaction were purified using Geneaid™ PCR purification kit (Geneaid) following the manufacturer’s protocol and cloned into pJET1.2/blunt vector (Fermentas). Then the vectors are introduced into Escherichia coli strain DH5α with heat shock method [47] . The plasmid vector was extracted by Geneaid™ plasmid isolation kit (Geneaid) and both strands were sequenced using pJET 1.2 forward and pJET 1.2 reverse primers at Macrogen Inc, South Korea.
Each of PCR products from single PCR reaction were purified using Geneaid™ PCR purification kit (Geneaid) following the manufacturer’s protocol and cloned into pJET1.2/blunt vector (Fermentas). Then the vectors are introduced into Escherichia coli strain DH5α with heat shock method [47] . The plasmid vector was extracted by Geneaid™ plasmid isolation kit (Geneaid) and both strands were sequenced using pJET 1.2 forward and pJET 1.2 reverse primers at Macrogen Inc, South Korea.
7
Comprehensive Genomic DNA Extraction and PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA extraction and polymerase chain reaction (PCR) amplification Each specimen was individually washed in sterile water and transferred into a 1.7 mL microcentrifuge tube. Total genomic DNA (gDNA) was extracted using the DNeasy® Blood & Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations.
PCR amplification was performed for 4 genetic markersthe mitochondrial COI, 12S rRNA, 16S rRNA genes and the nuclear ITS2 region. A T100™ thermocycler (Bio-Rad, California, USA) was used for amplification with a final PCR volume of 30 μL. Each reaction contained 15 μL of 2× i-Taq™ mastermix (iNtRON Biotechnology, Gyeonggi, South Korea), 5-10 μm of each primer and 2 μL of template DNA. The thermocycling profiles and primers used follow the protocol by Callejón et al. (2015a) for the COI gene and ITS2 region, while the protocols for the mitochondrial 12S and 16S rRNA genes follow Chan et al. (2020) . Amplicons were visualized on 1% agarose gel stained with SYBR™ Safe (Life Technologies, California, USA). Successful amplicons were purified with the Geneaid PCR Purification kit (Geneaid Biotech Ltd, Taipei, Taiwan) using the manufacturer's recommendations. The purified DNA samples were sent for Sanger sequencing by Macrogen (Seoul, South Korea) with the same primers used for PCR amplification.
PCR amplification was performed for 4 genetic markersthe mitochondrial COI, 12S rRNA, 16S rRNA genes and the nuclear ITS2 region. A T100™ thermocycler (Bio-Rad, California, USA) was used for amplification with a final PCR volume of 30 μL. Each reaction contained 15 μL of 2× i-Taq™ mastermix (iNtRON Biotechnology, Gyeonggi, South Korea), 5-10 μm of each primer and 2 μL of template DNA. The thermocycling profiles and primers used follow the protocol by Callejón et al. (2015a) for the COI gene and ITS2 region, while the protocols for the mitochondrial 12S and 16S rRNA genes follow Chan et al. (2020) . Amplicons were visualized on 1% agarose gel stained with SYBR™ Safe (Life Technologies, California, USA). Successful amplicons were purified with the Geneaid PCR Purification kit (Geneaid Biotech Ltd, Taipei, Taiwan) using the manufacturer's recommendations. The purified DNA samples were sent for Sanger sequencing by Macrogen (Seoul, South Korea) with the same primers used for PCR amplification.
8
Mitochondrial Gene Amplification and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amplification of mitochondrial 12S and 16S rRNA genes was performed in a thermocycler (Bio-Rad, California, USA) using the primers in Table 1 . PCR was performed in a final volume of 30 µl, containing 15 µl of 2× OnePCRTM master mix (GeneDireX. Inc., Taoyuan, Taiwan), 0.1 µM of each primer, and 5 ng/µl of DNA. The PCR thermocycling profiles were: 94 °C for 2 min of initial denaturation; then 35 cycles of 94 °C for 30 s, 45 °C to 55 °C for 1 min, and 72 °C for 2 min; followed by a final extension step at 72 °C for 5 min. The annealing temperatures used for each primer set are listed in Table 1 . Amplicons were checked and visualized on 1% agarose gel and stained with SYBRTM safe (Thermo Fisher Scientific, Waltham, USA). Successful amplicons were purified using the Geneaid PCR purification kit (Geneaid Biotech Ltd.). Sequencing for each sample was performed by a commercial company (Macrogen, Seoul, South Korea) on an automated Sanger sequencer. Mitochondrial 12S and 16S rRNA gene sequences generated in this study were deposited in the NCBI database (NCBI: MT135051-135093 for 12S rRNA; MT151872-151910 for 16S rRNA).
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!