The expression levels of RPL13A (a reference gene), VEE-hOct4, VEE-hKlf4, VEE-hSox2, VEE-hGlis1, Rex1, Oct4, and Nanog mRNA in ciPSCs were analyzed by RT-PCR. All the cDNA samples of ciPSC-like colonies were stored at −80 °C until PCR analysis. Total RNA was extracted from putative ciPSCs using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen) and random primers (Invitrogen). All the procedures were performed in accordance with the manufacturer’s instructions. RT-PCR analysis was conducted using cDNA from putative ciPSCs. cDNA was amplified in a 20 μL PCR mixture consisting of 10 pmol of forward and reverse primers, 2 units of Taq polymerase, 2 µL of 10x PCR buffer, 5 pmol of dNTP mixtures (all from iNtRON Biotechnology, SungNam, Korea), and template DNA. The oligonucleotide primer sequences are presented in Table S1 . PCR amplification was performed for 30 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 90 s. The reaction products were analyzed on a 1.25% agarose gel pre-stained with RedSafe™ Nucleic Acid Staining Solution (iNtRON Biotechnology).
Redsafe nucleic acid staining solution
Manufactured by iNtRON Biotechnology
Sourced in United States, Cameroon, Canada
RedSafe Nucleic Acid Staining Solution is a nucleic acid stain used for the detection and visualization of DNA and RNA in agarose gels. It is a fluorescent dye that binds to nucleic acids and emits a bright red fluorescence when exposed to ultraviolet (UV) light.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Takara Bio (3 mentions)
Qiaamp viral rna mini kit, by Qiagen (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Cdna master mix, by Toyobo (2 mentions)
Rnaiso plus reagent, by Takara Bio (2 mentions)
Gel doc xr system, by Bio-Rad (2 mentions)
Max enteric viral panel, by BD (2 mentions)
Max system, by BD (2 mentions)
Image quant tl analysis software, by Cytiva (2 mentions)
Easy blue total rna extraction kit, by iNtRON Biotechnology (2 mentions)
Concentrator plus, by Eppendorf (2 mentions)
G spin total dna extraction kit, by iNtRON Biotechnology (2 mentions)
Matrigel matrix, by BD (2 mentions)
Rna extraction kit, by Bioneer (2 mentions)
Topsimple dyemix ntaq, by Enzynomics (2 mentions)
Nucleospin gel and pcr clean up, by Macherey-Nagel (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
72 protocols using redsafe nucleic acid staining solution
1
Characterization of Induced Pluripotent Stem Cells
2
SRAP Primers for Banana Polymorphism
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A set of 11 SRAP primers (Table 6) were designed by Li and Quiros (2001) and used to search for polymorphism among treated and untreated banana plants. The total reaction mixture was 25 µl contained 10X PCR buffer, 2 mM MgCl 2 , 0.2 mM dNTPs mixed, 10 pmol primers, 1.25 U Taq polymerase and about 150 ng genomic DNA. The amplification regime followed the recommendation of Li and Quiros (2001) as follows: An initial denaturing step was performed at 94 °C for 5 min, followed by 5 cycles at 94 °C for 1 min, 35 °C for 1 min and 72 °C for 1 min, subsequently followed by 35 cycles at 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min, the final extension step at 72 °C for 7 min.
Amplification products were separated on a 1.5% agarose gel containing 1X TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) at 90 V. The genomic DNA was stained with RedSafe Nucleic Acid Staining Solution (1/20,000) (iNtRON Biotechnology, Inc. Kr). Gels were analyzed by UVI Geltec version 12.4, 1999 -2005 (USA) .
Amplification products were separated on a 1.5% agarose gel containing 1X TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) at 90 V. The genomic DNA was stained with RedSafe Nucleic Acid Staining Solution (1/20,000) (iNtRON Biotechnology, Inc. Kr). Gels were analyzed by UVI Geltec version 12.4, 1999 -2005 (USA) .
3
Lactobacillus Strain Identification via 16S rRNA PCR
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The DNA was extracted from the isolated bacteria and Lactobacillus strains were confirmed by using 16S rRNA multiplex PCR analysis (Kwon et al., 2004) (link). The reaction mixture (25 μl) contained 12.5 μl of PCR Master Mix, 5 μl primer mixture comprising 50 pmol of each primer, 4.5 μl of water, and 3 μl of DNA template. PCR amplification was performed in Applied Biosystem 2720 thermal cycler, and DNA fragments were amplified as follows. Initial heating at 94 °C for 2 min, 35 cycles consisting of denaturation at 94 °C for 20 sec, annealing at 51 °C for 40 sec, extension at 68 °C for 30 sec, and final extension step in 7 min at 68 °C. The PCR products were separated on 1.5% agarose gel by electrophoresis and analyzed by RedSafe Nucleic Acid Staining Solution (Intron Biotechnology, Korea).
4
Cercarial ITS2 Amplification and Sequencing
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About 1% of the samples of each cercarial group was randomly selected and used for molecular analysis. Total genomic DNA was isolated from ethanol-fixed cercariae using proteinase K (10 mg/ml) (Derycke et al. 2012) (link) and Worm Lysis Buffer (WLB) (Williams et al. 1992 ) and stored at -2 0 °C . T h e f o r w a r d p r i m e r I T S 3 ( 5 ′ -G C A T CGATGAAGAACGCAGC-3′) and the reverse primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) targeting the cercarial ITS2 region were used following Barber et al. (2000) . The amplicons were visualized on 1% agarose gel (1 g agarose per 100 ml TAE buffer 0.5X) stained with 4 μl RedSafe™ Nucleic Acid Staining Solution (iNtRon Biotechnology). The PCR products were purified, and cycle sequencing reactions based on Sanger method were performed (in both directions) by BigDye® Terminator v3.1 at 1st BASE company (Malaysia).
5
Quantification of Osteoblastic Gene Expression
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The expressions of RANKL, OPG, TRPM3, TRPV4 and betaactin were evaluated by RT-PCR using total RNA isolated from murine osteoblastic cells. Cells were lysed in TRIzol reagent according to the instructions of the manufacturer (Invitrogen). AccuPower RT PreMix (BIONEER, Daejeon, Korea) with total RNA (1 µg) was used for cDNA synthesis. cDNA was amplified by PCR with HiPi Thermostable DNA polymerase (Elpis, Pusan, Korea) using the following primer sets: RANKL (forward): 5′-ATCAGAAGACAGCACTCACT-3′ (reverse): 5′-ATCTAGGACATCCATGCTAATGTTC-3′; OPG (forward): 5′-TGAGTGTGAGGAAGGGCGTT-3′ (reverse): 5′-TTCCTCGTTCTCTCAATCTC-3′; TRPM3 (forward): 5′-CACCTGATGACCAAGGAATG-3′ (reverse): 5′-CTTGTG TTTATCTTCTGGAGTG-3′; TRPV4 (forward): 5′-GCTGAA GGCAAAAGTCTTGG-3′ (reverse): 5′-CTAGGGAACCCCAA CTGTGA-3′; beta-actin (forward): 5′-TGTGATGGTGGG AATGGGTCAG-3′ (reverse): 5′-TTTGATGTCACGCACG ATTTCC-3′. PCR was performed under the following conditions: 94°C for 5 min, 94°C for 30 s, 40 s for annealing step (temperatures varied with primers), 72°C for 30 s, followed by 72°C for 10 min after 38 cycles were finished. The annealing temperature was 60°C for TRPM3, 56.5°C for TRPV4 and RANKL and 57.6°C for OPG and beta-actin. The PCR products were separated on 1.2% agarose gels and visualized with RedSafe nucleic acid staining solution (iNtRon Biotechnology, Gyeonggi-do, Korea).
6
Microbial 16S rRNA Sequencing and Identification
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The bacteria with most interesting properties were selected for PCR amplification of conserved 16S rRNA and subsequent sequencing and analysis. For PCR amplification, universal primers 16S F8-27 (5′-AGAGTTTGATCCTGGCTCAG-3′) and 16S R1541-1522 (5′-AAGGGAGGTGATCCAGCCGCA-3′) were employed. The reaction mixture contained for 25 μl: 10x Ecogen buffer 2.5 μl, MgCl2 50 mM 1 μl, dNTPs 10 mM (2.5 mM each) 1 μl, EcoTaq polymerase (5U/μl) 0.2 μl, extracted DNA 1 μl, each primer 10 μM 1 μl and H2OMQ17.3 μl. The temperature profile was programmed as follows: predenaturation at 95°C for 2 min, 35 cycles of denaturation at 95°C for 45 s, annealing at 58°C for 45 s, extension at 72°C for 90 s (35 cycles) and final extension at 72°C for 5 min. The amplified products were checked by running on 1% agarose gel and visualized under UV after staining with RedSafe™ Nucleic Acid Staining Solution (iNtRON Biotechnology, Korea). The PCR product was purified with SpeedTools PCR Clean-up kit (Biotools, Spain) and sequencing was done by StabVida Company (Portugal). The EzTaxon server was used to determine 16S rRNA sequence homologies (Chun et al., 2007 (link)). Finally, accession numbers from KT036396 to KT036409 were assigned to the sequences deposited in GenBank (Table 2 ).
7
Quantifying Apoptosis Markers in Cell Lines
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Total RNA was extracted from YCC-2 and SNU-668 cells treated with G. thunbergii using RNAiso Plus reagent (TaKaRa Bio, Japan), and cDNA was synthesized using cDNA Master Mix (ToYoBo, Japan) according to the manufacturer’s protocol. Reverse transcription PCR was performed as described previously (Kim et al. 2018 (link)) using the following PCR primer sequences: Bcl-2, 5ʹ-GGATGCCTTTGTGGAAAACCCTGT-3ʹ (forward) and 5ʹ-AGCCTGCAGCTTTGTTTCAT-3ʹ (reverse); Bax, 5ʹ-TTTGCTTCAGGGTTTCATCC-3ʹ (forward) and 5ʹ-CAGTTGAAGTTGCCGTCAGA-3ʹ (reverse); caspase-3, 5ʹ-TTTTTCAGAGGGGATCGTTG-3ʹ (forward) and 5ʹ-CGGCCTCCACTGGTATTTTA-3ʹ (reverse); PARP, 5ʹ-TGGAACATCAAGGACGAGCT-3ʹ (forward) and 5ʹ-GCATCGCTCTTGAAGACCAG-3ʹ (reverse); GAPDH, 5ʹ-GGCTGCTTTTAACTCTGGTA-3ʹ (forward) and 5ʹ-ACTTGATTTTGGAGGGATCT-3ʹ (reverse). PCR products were used for 1% agarose gel electrophoresis with RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology).
8
PCR Detection of Biofilm Genes
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PCR detection of biofilm-associated genes (bap, csuE, ompA, adeFGH, abaI) was performed on a Veriti thermocycler (Applied Biosystems) using sets of primers shown in Table S1 . PCR was performed in a 20-µL reaction consisting of 1 µL extracted genomic DNA, 10 µL Luna Universal qPCR Master Mix (New England Biolabs, USA), 1 µL (10 µM) of each primer. The reaction condition was initial denaturation at 95 °C for 7 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 1 min and extension at 72 °C for 30 s, and a final extension of 72 °C for 10 min. PCR products were separated by 1.5% agarose gel electrophoresis the gel was stained with 0.5 μg/mL RedSafe nucleic acid staining solution (iNtRON Biotechnology, Korea). The stained gel was visualized using a Gel Doc System XR (Bio-Rad Laboratories).
9
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated using TRIZOL reagent (Takara Bio, Inc., Tokyo, Japan) according to the manufacturer's instruction. Complementary DNA (cDNA) was synthesized by reverse transcriptase reaction. PCR amplification was performed with specific primers using thermal cycler SimpliAmp, Thermo Fisher Scientific, MA, USA). The products were run on 2% agarose gel (Bioneer Co., Daejeon, Korea) in 1X Tris Acetate-EDTA (TAE) buffer (Elpis Biotech, Daejeon, Korea) containing RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, Seongnam, Korea). Sequences of primers used for RT-PCR: Olig2 forward 5′-GAATCCGCTGGTATCCACGA-3′, Olig2 reverse 5′-GCGGAGCGAGACGTTTAGAA-3′, MMP-2 forward 5′-GGCCCTGTCACTCCTGAGAT-3′, MMP-2 reverse 5′-GGCATCCAGGTTATCGGGGA-3′, MMP-9 forward 5′-GATGCGTGGAGAGTCGAAAT-3′, MMP-9 reverse 5′- CACCAAACTGGATGACGATG-3′, β-actin forward 5′-GGCATCGTGATGGACTCCG-3′, β-actin reverse 5′- GCTGGAAGGTGGACAGCGA-3’.
10
Molecular Surveillance of Malaria Drug Resistance
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The drug resistance marker genes Pfcrt (PF3D7_0709000), Pfdhfr (PF3D7_0417200), Pfdhps (PF3D7_0810800), Pfk13 (PF3D7_1347700), and Pfmdr1 (PF3D7_0523000) and ama1 (PF3D7_1133400) from the DNA reference isolates were amplified using nested PCR. The first PCR assay was set up as a 10-µL final reaction volume as follows: 1 µL of template DNA (<50 ng), 0.14 µL Expand high-fidelity DNA polymerase (3.5 U/µL) (Roche, USA), 0.3 µL forward and reverse 10 mM external primers (Table S1), 0.2 µL of 10 µM deoxynucleoside triphosphates (dNTPs) (Bioline), 1 µL each of buffers 2 and 4, and 6.56 µL of nuclease-free water. The nested PCR was prepared as described above, except that 1 µL of the first PCR assay product with multiplex identifier (MID) (Roche, USA)-tagged (Table S2) internal forward primers and untagged reverse internal primers (Table S1) were used. Each sample was amplified in duplicate with nonoverlapping MID tags. The following PCR cycling conditions were used: 94°C for 2 min, 10 cycles of 94°C for 15 s, 52°C for 30 s, and 72°C for 45 s, followed by an additional 20 cycles of 94°C for 15 s, 52°C for 30 s, and 72°C for 45 s and a final elongation step of 72°C for 5 min. Successful PCR amplification was confirmed using 1% (wt/vol) agarose gels stained with RedSafe nucleic acid staining solution (iNtRON Biotechnology DR).
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