The extracted RNA was treated with DNase to eliminate any contamination. One µL of random primers, mainly random hexanucleotides (500 µg mL−1; Promega Corporation, Madison, WI, USA) were added. A conversion mix (M-MLV RT 5×, M-MLV RT transcriptase, RNasin and dNTPs) was added. The first PCR program (MultiGene optiMAX; Labnet International Inc., Edison, NJ, USA) was: 5 min at 20 °C; 45 min at 37 °C; 5 min at 95 °C; and 4 °C until storage. A PCR using the primers for 18S rRNA subunit (Table S3, Supplemental Material ) was performed to verify the correct conversion. Conditions of the second PCR program were: 3 min at 95 °C; 30 cycles of 1 min each at 94 °C followed by 1 min at 61 °C, and 1 min at 72 °C; 10 min at 72 °C; and 4 °C until storage. The cDNA was stored for validation assays.
Multigene optimax
Manufactured by Labnet
Sourced in United States
The MultiGene Optimax is a compact and versatile thermal cycler designed for DNA amplification and analysis. It features a high-performance Peltier heating/cooling system, a large graphical LCD display, and intuitive user interface for easy operation. The MultiGene Optimax can accommodate up to 96 samples in 0.2 mL tubes or 384-well plates.
Automatically generated - may contain errors
Lab products found in correlation
Agarose gel, by Bioneer (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Random hexanucleotide, by Promega (1 mentions)
Firepol master mix, by Solis BioDyne (1 mentions)
T1 thermal cycler, by Analytik Jena (1 mentions)
Green gotaq reaction buffer, by Promega (1 mentions)
Tae buffer, by Bioneer (1 mentions)
Mgcl2, by Promega (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Uv transilluminator, by Vilber (1 mentions)
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
Abi 3730 dna sequencer, by Thermo Fisher Scientific (1 mentions)
Genejet genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Phusion high fidelity polymerase, by Thermo Fisher Scientific (1 mentions)
Blood and tissue kit, by Qiagen (1 mentions)
Hf buffer, by Thermo Fisher Scientific (1 mentions)
Mytaq polymerase, by Meridian Bioscience (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Nanodrop nd 100, by Thermo Fisher Scientific (1 mentions)
18 protocols using multigene optimax
1
RNA Extraction and Reverse Transcription
2
Profiling Virulence Genes in Helicobacter pylori
3
RAPD Analysis of DNA Samples
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RAPD analysis was performed by using 10-mer primer from Operon Technologies (Alameda, CA, USA). Nine arbitrary RAPD primers were tested for amplification (Table 2). DNA amplification was performed in 20 µl reaction volume containing 1.5 mM MgCl2, 10 mM dNTPs, 0.8 picomole of primer, 1.5 U Taq DNA polymerase and 50 ng template DNA. PCR amplification was performed in a Thermo cycler (MULTIGENE OPTIMAX, Labnet International, Inc.) with the following cycling conditions: denaturation at 94ºC for 1 min, annealing at 37ºC for 1 min and elongation at 72ºC for 2 min for 40 cycles after an initial denaturation for 5 min at 94ºC. After finishing of amplification 12 µl aliquots of amplification products were loaded in a 1% (w/v) agarose gel (Bioneer) for electrophoresis in 1X TAE buffer (Bioneer). Gels were stained with Ethidium bromide (0.5 µg/ml for 30 min) and visualized under exposure of UV light within gel doc system (UVDI, Major Science). There was standard 1 kb ladder (Promega) for comparing band size.
4
SSR-Based DNA Profiling Protocol
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Eight pairs of SSR primers were tested for amplification and among them only five primers were found good for DNA profiling (Table 2) based on readable and reproducibly of the bands. SSR amplification was performed in 20 µl reaction volume containing 50ng genomic DNA, 10 µl of 2X Green GoTaq® Reaction Buffer (pH 8.5), 400μM dATP, 400μM dGTP, 400μM dCTP, 400μM dTTP and 3 mM MgCl2 (Promega) and 10 picomole of each forward and reverse primer. These components were gently mixed and centrifuged prior to adding 2 drops of mineral oil. The amplification was performed in a Thermo cycler (MULTIGENE OPTIMAX, Labnet International, Inc.). The cycling conditions were 1 cycle of 95°C for 5 min followed by 30 cycles of 95°C for 60 sec, 55°C for 1 min 30 sec, 72°C for 2 min, and finally 1 cycle of 72°C for 10 min.
After finishing amplification 12 µl aliquots of amplification products was loaded in a 1% (w/v) agarose gel (Bioneer) for electrophoresis in 1X TAE buffer (Bioneer). After completion of electrophoresis, gels were stained with Ethidium bromide (0.5 µg/ml for 30 min) and visualized under exposure of UV light within gel doc system (UVDI, Major Science). There was standard 100 bp ladder (Promega) for comparing size of the band.
After finishing amplification 12 µl aliquots of amplification products was loaded in a 1% (w/v) agarose gel (Bioneer) for electrophoresis in 1X TAE buffer (Bioneer). After completion of electrophoresis, gels were stained with Ethidium bromide (0.5 µg/ml for 30 min) and visualized under exposure of UV light within gel doc system (UVDI, Major Science). There was standard 100 bp ladder (Promega) for comparing size of the band.
5
Genomic DNA Extraction and 16S rRNA Amplification
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The salting-out method was followed to extract the total genomic DNA of the most potent ACP-producing isolate. Afterward, the amplification of the fragments of the 16S rRNA gene by the PCR reaction, using universal forward primer 5′-AGAGTTTGATCMTGGCTCAG-3′ (corresponded to position 8 of Escherichia coli16S rRNA) and reverse primer 3′-TACGGYACCTTGTTACGACTT-5′ (corresponded to position 1514 of Escherichia coli16S rRNA). The PCR reaction mixture consisted of (1.5 μl) 10 pM for each primer, 0.1 μg of chromosomal DNA, (5 μl) 2 mM dNTPs, (0.4 μl) 2U of Taq polymerase, (5 μl) 1 × polymerase buffer (Fermentas, Germany) and 36.8 μl of nuclease-free water for a 50 μl reaction. PCR amplification was performed using a thermal cycler (Multigene Optimax, Labnet International, Inc.) under the following conditions: 4.0 min at 95 °C, then 30 cycles at 94 °C for 1.0 min, 55 °C for 1.0 min, and 72 °C for 2.0 min, followed by an additional 10 min at 72 °C as a final extension step. Sequencing, and the construction of a neighbor-joining phylogenetic dendrogram, were executed according to the method of Abdelgalil et al.42 (link).
6
ISSR Marker Amplification Protocol
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We used 18 UBC (University of British Columbia) markers for the ISSR amplification: UBC802, UBC807, UBC808, UBC809, UBC810, UBC811, UBC813, UBC815, UBC816, UBC818, UBC821, UBC823, UBC825, UBC834, UBC850, UBC880, UBC856 and ISSR001 [53 (link),54 (link)], of which 12 were selected that provided higher numbers of fragments and better reproducibility. The 20 µL PCR solution consisted of: 10 µL Master Mix SapphireAmp 2× (Takara, Clontech, USA), 5 µL ISSR primer (5 µM), 1 µL genomic DNA (5 ng/µL) and 4 µL nuclease-free water. Amplifications were performed in a MultiGene Optimax thermal cycler (Labnet) using the following protocol: an initial step of 5 min at 94 °C, 45 cycles of 30 s at 94 °C, 45 s at 52 °C and 2 min at 72 °C, followed by a final extension step of 6 min at 72 °C. The amplification products were separated by agarose gel electrophoresis (1.2%) and colored with GelRedTM (10,000×) at the ratio of 10 µL/100 mL of gel (with Tris-borate-EDTA at 0.5×). The electrophoresis run was performed at 100 V for 110 min. The gel was then placed in a UV trans illuminator (Vilber Lourmat, France) and photographed with a digital camera (Canon, SX160 IS. USA).
7
Amplification and Sequencing of trnL Intron
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For the amplification of the trnL intron (UAA) the primers forward A49325 5′-CGAAATCGGTAGACGCTACG-3′ and reverse B49863 5′-GGGATAGGGACTTGAAC-3′ described by Taberlet et al. [21 (link)] were used. The 24 µL PCR solution was prepared as follows: 12 µL Master Mix SapphireAmp 2× (Takara, Clontech), 1.5 µL forward (5 µM), 1.5 µL reverse (5 µM), 6 µL genomic DNA (5 ng/µL) and 3 µL nuclease-free water. Amplification was performed in a MultiGene Optimax thermal cycler (Labnet) using the following conditions: an initial step of 5 min at 95 °C, 35 cycles of 45 s at 95 °C, 45 s at 55 °C and 2 min at 72 °C, followed by a final step of extension of 6 min at 72 °C. The amplified products were separated and visualized as described in the “ISSR Amplification” section. The PCR amplicons were cut from the gel and purified with the Wizard® SV Gel and PCR Clean-Up System (Promega) kit, according to the manufacturer’s recommendations. The purified PCR product was then shipped to Macrogen Inc. for sequencing using ABI3730XL equipment. (Seoul, South Korea).
8
16S rRNA Gene Amplification and Sequencing
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DNA concentration was measured with a spectrophotometer (Jenway, Staffordshire, OSA, UK). DNA was amplified by polymerase chain reaction (PCR) (Multigene Optimax, Labnet International, Inc., Edison, NJ, USA) using suitable primers; the forward primer was 5ʹ-AGAGTTTGATCMTGGCTCAG-3ʹ (position 8 in the 16S rRNA gene according to E. coli numbering) and the reverse primer was 3ʹTACGGYACCTTGTTACGACTT-5ʹ (position 1514 in the 16S rRNA gene according to E. coli numbering). After gel electrophoresis to confirm the success of the previous steps, the amplified DNA was stored in nuclease-free water and sequenced using an ABI 3730 DNA sequencer (Thermo Fisher Scientific, Waltham, MA, USA).
9
Genotyping of Akt SNP via PCR
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DNA was extracted using DNA extraction kit (GeneJET Genomic DNA Purification Kit, Thermo Scientific, USA) and was quantified using UV-visible spectrophotometer (752 PC, China). Akt single nucleotide polymorphism was determined using polymerase chain reaction (PCR, Multigene Optimax, Labnet International, USA). PCR was performed in a 20 μL reaction mixture using allele specific primers. The sequences of primers and amplification condition are given in Table 1 . The AA (379 bp), AG (245 and 379 bp) and GG (245 bp) genotypes were visualized with ethidium bromide and identified on agarose gel (2%) using UV transilluminator (Wealtec, USA).
10
ISSR-Based Genotyping Protocol
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The DNA amplification was done with a 25 μL final PCR reaction volume for each primer (12.5 μL og GoTaq® Green Master Mix (Promega CO) 0.5 μL primer, 10 μL nuclease-free water, and 2 μL DNA). The final primer concentration for amplification was 0.25 μM, as suggested by Medina-Medrano et al. (2016) and Latorre et al. (2013) . The PCR was done in a MultiGene™ OptiMax (Labnet) thermocycler, and the thermal conditions were: initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 60 s, 49 -64°C for 120 s depending on the ISSR primer (Tab. 1), 120 s at 72°C and a final extension at 72°C for 7 min. DNA Amplicons were confirmed in a high resolution 2% agarose gel at 80V for 2 h, stained with SYBR Green and later visualized in a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Hercules, USA). The gel-band pattern was used to assign the genotype profile for every ramet.
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