The most MDR strains from the Escherichia, Klebsiella, and Pseudomonas genera were selected for identification using 16S rRNA partial gene sequencing. The selected strains were identified using genomic DNA isolation, amplification, sequencing of nucleotides, and phylogenetic analysis. The bacterial biomass was treated with proteinase-K treatment for the isolation of genomic DNA.28 (link) For PCR, 2.5 μL genomic DNA was used. The PCR reaction was performed using primers 27F 5' (AGA GTT TGA TCM TGG CTC AG) 3′ and 1492R 5' (TAC GGY TAC CTT GTT ACG ACT T) 3'. The amplified PCR product was run on 1% agarose gel with GeneRuler 1 kb DNA (Fermentas) to confirm the size of the amplified 16S rRNA and further purified using a PCR purification kit (Favorgen, Taiwan). The amplified PCR products were sequenced using the commercial services of MACROGEN Seoul, Korea (http://macrogen.com/eng/ ). The resulting nucleotide sequences were blasted on NCBI servers by optimising highly similar sequences (Megablast). The sequences of closely related strain types were selected, and the phylogenetic tree was drawn using MEGA 7.0.14.29 (link)
Pcr purification kit
Manufactured by Favorgen Biotech
The PCR purification kit is a laboratory tool designed to purify DNA fragments amplified by PCR (Polymerase Chain Reaction). It is used to remove unwanted components, such as primers, nucleotides, and enzymes, from the PCR reaction mixture, allowing for the isolation of the desired DNA product.
Automatically generated - may contain errors
Lab products found in correlation
Ef taq premix, by Solgent (3 mentions)
3500 dna sequencer, by Thermo Fisher Scientific (2 mentions)
Exprep plasmid sv kit, by GeneAll (1 mentions)
Abi 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Ecodye, by Biofact (1 mentions)
Gel doc, by Bio-Rad (1 mentions)
Catechol, by HiMedia (1 mentions)
Potassium ferrocyanide, by HiMedia (1 mentions)
4 aminoantipyrine, by HiMedia (1 mentions)
K2cr2o7, by Merck Group (1 mentions)
Taq dna polymerase master mix, by Ampliqon (1 mentions)
Cdcl2, by Merck Group (1 mentions)
Znso4 h2o, by Merck Group (1 mentions)
Cocl2.2h2o, by Merck Group (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Phenol, by Merck Group (1 mentions)
Hgcl2, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by HiMedia (1 mentions)
9 protocols using pcr purification kit
1
Microbial Identification via 16S rRNA Sequencing
2
Validation of Candidate Loci by PCR and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate candidate loci, we conducted PCR amplification and Sanger sequencing. A primer pair for the PCR amplification was designed in the flanking region of each target locus using OligoCalc (http://www.basic.northwestern.edu/biotools/oligocalc.html ) and Oligo Analysis Tools (http://www.operon.com/tools/oligo-analysis-tool.aspx ). Detailed information on the primers is summarized in Supplementary Table 3 . PCR was performed in 20 µL of the reaction mixture, including 10 µL of 2× EF-Taq Pre mix4 (Biofact, Daejeon, Korea), 10 µM of oligonucleotide primers, 1 µL of template, and nuclease-free water. PCR was carried out as follows: first denaturation step of 3 min at 95℃, followed by 25 cycles of 30 s at 95℃, annealing of 30 s at optimal temperature, extension of 40 s to 1 min depending on PCR product size at 72℃, and a final extension for 2 min at 72℃. PCR products were confirmed by gel electrophoresis and purified with a PCR purification kit (Favorgen Biotech Corp., Pingtung Country, Taiwan). Products were sequenced using an ABI3500 genetic analyzer (Thermo Fisher Scientific, Pittsburgh, PA, USA). Sequencing data were aligned with exome sequencing data by the Bioedit program.
3
PCR Amplification and Sequencing of SNPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polymerase chain reaction (PCR) amplification of two SNPs was performed at 95℃ for 10 minutes, followed by 35 cycles at 95℃ for 30 seconds, 55℃~58℃ for 30 seconds and 72℃ for 40 seconds, with a final extension at 72℃ for 90 seconds. The PCR reaction mixtures (total volume 50 µl) contained 25 µl of 2X EF-Taq premix (SolGent, Seoul, Korea), 18 µl of distilled water, 2.5 µl of oligonucleotide primer (10 pmol/µl), and 2 µl of template containing 20 ng genomic DNA. The PCR products were purified using a PCR purification kit (Favorgen, Pingtung, Taiwan) and were sequenced on an Applied Biosystems 3500 DNA sequencer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.
4
Genomic DNA PCR Amplification and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR was performed using two different human genomic DNA samples (KPGP9 and NA10851 (Coriell Cell Repository, USA)) as templates. PCR amplification of each locus was performed in 20 ul reactions containing 20 ng of template DNA, 10 ul of 2X EF-Taq Premix4 (BioFACT), 10 nM of each oligo nucleotide primers, and nuclease-free water. Each PCR was subjected to initial denaturation step of 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 40 s of annealing at optimal annealing temperature, and a long extension step at 68°C for 7 min, followed by a final extension step at 68°C for 10 min. The PCR products were run on a 1% agarose gel electrophoresis with EcoDye (BioFACT) and visualized using Gel Doc (Bio-Rad, Germany). The PCR product was purified by the PCR purification kit (FAVORGEN, Taiwan) and some products were cloned with the Dr. TA TOPO cloning kit (Doctor Protein, Korea) according to the manual instructions. Target clone were confirmed by colony PCR, following plasmid preparation with the Exprep™ plasmid SV kit (GeneAll Biotechnology, Korea) and sequenced using chain-termination sequencing on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA).
5
Sanger Validation of Three SNPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three SNPs were selected for Sanger validation. PCR amplification of all SNPs was performed at 95 °C for 10 min, followed by 35 cycles at 95 °C for 30 s, 55–58 °C for 30 s and 72 °C for 40 s, with a final extension at 72 °C for 1 min 30 s. The PCR mixtures (total volume 50 μL) contained 25 μL of 2X EF-Taq premix (SolGent, Seoul, South Korea), 2.5 μL of oligonucleotide primer (10 pmol/μL), 18 μL of distilled water, and 2 μL of template containing 20 ng genomic DNA. The PCR products underwent purification via a PCR purification kit (Favorgen, Pingtung, Taiwan) and were sequenced on an Applied Biosystems 3500 DNA sequencer (Foster City, CA, USA) according to the manufacturer’s instructions.
6
PCR Amplification and SNP Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polymerase chain reaction (PCR) amplification of two single nucleotide polymorphisms (SNPs) was performed at 95℃ for 10 minutes, followed by 35 cycles at 95℃ for 30 seconds, 55℃~58℃ for 30 seconds and 72℃ for 40 seconds, with a final extension at 72℃ for 1 minute 30 seconds. The PCR reaction mixtures (total volume 50 µl) contained 25 µl of 2X EF-Taq premix (SolGent, Seoul, Korea), 18 µl of distilled water, 2.5 µl of oligonucleotide primer (10 pmol/µl), and 2 µl of template containing 20 ng genomic DNA. The PCR products were purified using a PCR purification kit (Favorgen, Pingtung, Taiwan) and were sequenced on an Applied Biosystems 3500 DNA sequencer (Foster City, CA, USA) according to the manufacturer's instructions.
7
Sanger Sequencing of MYBPC3 Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The amplified PCR product was subsequently purified by PCR Purification Kit (Favorgen, Ping-Tung, Taiwan) for DNA sequencing. The sequencing reaction was conducted with the Sanger sequencing method, using the same specific primers as mentioned above. The DNA sequencing for MYBPC3-A74T and A31P polymorphism was analyzed with the Bioedit program.
8
PCR Amplification and DNA Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polymerase chain reaction (PCR) amplification conditions were as follows: 95℃ for 10 minutes, followed by 35 cycles at 95℃ for 30 seconds, 55℃~58℃ for 30 seconds, and 72℃ for 40 seconds, with a final extension at 72℃ for 1 minutes 30 seconds. The PCR mix (50 µl) contained 25 µl of 2× EF-Taq premix (SolGent, Seoul, Korea), 18 µl of distilled water, 2.5 µl of oligonucleotide primer (10 pmol/µl), and 2 µl of template containing 20 ng genomic DNA. The PCR products were purified, using a PCR purification kit (Favorgen, Pingtung, Taiwan), and sequenced on an Applied Biosystems 3500 DNA sequencer (Applied Biosystems, Foster City, CA, USA), according to the manufacturers' instructions.
9
Heavy Metal Tolerance Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Davis minimal medium (DMM), 4-aminoantipyrine, potassium ferrocyanide, catechol, sodium hydroxide pellets used in this study were purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, phenol, 2-mercaptoethanol, ammonium hydroxide, glycerol, disodium EDTA, primers, acids and solvents were procured from Sigma, Aldrich, Tris Base, Agarose from Bio Basic Inc. All the following heavy metals CoCl2.2H2O, ZnSO4.H2O, (CH3COO)2Pb.3H2O, HgCl2, CdCl2, CuSO4.5H2O, NiCl2.6H2O, MnCl2.4H2O, and K2Cr2O7 were purchased from Merck Millipore, Taq DNA polymerase Master Mix from Ampliqon and PCR purification Kit from Favorgen Biotech Corp. were used.
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!