Gel electrophoresis was conducted to confirm the on-chip amplification as follows. A 2% agarose (Bioneer) solution was prepared by microwaving with 30 s pulses for 3 min. A 2 μL aliquot of 10 mg/mL ethidium bromide (EtBr) (Sigma-Aldrich) was added to 50 mL of a 2% agarose solution to visualize the DNA under UV light, and the mixture was solidified in a template. The prepared gel was placed into the gel chamber and filled with 1× TBE (Bioneer) containing 5 μL of 10 mg/mL EtBr. A 2 μL aliquot of eluted RNA from the chip was mixed with 6 μL of loading buffer (Sigma-Aldrich) and loaded carefully along with the DNA ladder (Sigma-Aldrich) into the lanes of the gel. Gel electrophoresis was conducted at 80 V for 1 h until the dye line was approximately 80% of the way down the gel. The gel was carefully placed inside a UV box (Gel documentation system, GDS-200D) to take a photographic image.
Agarose
Manufactured by Bioneer
Sourced in United States
Agarose is a polysaccharide derived from red seaweed. It is commonly used as a gel medium in various laboratory applications, particularly in electrophoresis techniques. Agarose gels are known for their ability to separate and analyze biomolecules such as proteins and nucleic acids based on their size and charge.
Automatically generated - may contain errors
Lab products found in correlation
Dna ladder, by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Loading buffer, by Merck Group (1 mentions)
100 bp dna ladder, by Bioneer (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Isopropyl alcohol, by Merck Group (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Eosin y, by Merck Group (1 mentions)
Folin ciocalteu s reagent, by Merck Group (1 mentions)
Ethanol, by Daejung Chemicals (1 mentions)
Rt pcr premix kit, by Bioneer (1 mentions)
Chloroform, by Daejung Chemicals (1 mentions)
Magellan 400, by Thermo Fisher Scientific (1 mentions)
4 protocols using agarose
1
Gel Electrophoresis for On-Chip Amplification Validation
2
Genetic Polymorphism Analysis of Gracilaria Species
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The oligonucleotides primer selected were purchased from Operon Technologies,
USA. The G + C content of the primers is between 60%–70%. Seven
oligonucleotides primers, OPC-01 (5’-TTCGAGCCAG-3’), OPA-04
(5’-AAT CGGGCTG-3’), OPA-05 (5’-AGGGGTCTTG-3’),
OPD-07 (5’-TTGGCACGGG-3’), OPD-08 (5’-GTGTGCCCCA-
3’), OPB-10 (5’-CTGCTGGGAC-3’) and OPD-16 (5’-AG
GGCGTAAG-3’) were shown to generate the unique shared loci to each
species and shared loci by the two species that can be scored clearly and
reproducibly. Thus, we used the oligonucleotides primers to identify the genetic
polymorphism, diversity and similarity of two Gracilariaspecies. PCR technique was performed using two programmable DNA thermal cyclers
(Perkin Elmer Cetus, USA; MJ Research, Inc., USA). The DNA amplification was
performed in 25 µL containing 10 ng of template DNA, 20 µL premix
(Bioneer Co., Daejeon, Korea) and the 1.0 unit primer. Amplification products
were separated by 1.4% agarose (Bioneer Co., Daejeon, Korea) gel electrophoresis
with TBE (90 mM Tris, pH 8.5; 90 mM borate; 2.5 mM EDTA). The 100 bp DNA Ladder
(Bioneer Co., Daejeon, Korea) was used as DNA molecular weight marker. The
agarose gels electrophoresed were stained with ethidium bromide. The fragments
were illuminated with ultraviolet ray and then photographed by photoman direct
copy system (PECA products, Beloit, WI, USA).
USA. The G + C content of the primers is between 60%–70%. Seven
oligonucleotides primers, OPC-01 (5’-TTCGAGCCAG-3’), OPA-04
(5’-AAT CGGGCTG-3’), OPA-05 (5’-AGGGGTCTTG-3’),
OPD-07 (5’-TTGGCACGGG-3’), OPD-08 (5’-GTGTGCCCCA-
3’), OPB-10 (5’-CTGCTGGGAC-3’) and OPD-16 (5’-AG
GGCGTAAG-3’) were shown to generate the unique shared loci to each
species and shared loci by the two species that can be scored clearly and
reproducibly. Thus, we used the oligonucleotides primers to identify the genetic
polymorphism, diversity and similarity of two Gracilariaspecies. PCR technique was performed using two programmable DNA thermal cyclers
(Perkin Elmer Cetus, USA; MJ Research, Inc., USA). The DNA amplification was
performed in 25 µL containing 10 ng of template DNA, 20 µL premix
(Bioneer Co., Daejeon, Korea) and the 1.0 unit primer. Amplification products
were separated by 1.4% agarose (Bioneer Co., Daejeon, Korea) gel electrophoresis
with TBE (90 mM Tris, pH 8.5; 90 mM borate; 2.5 mM EDTA). The 100 bp DNA Ladder
(Bioneer Co., Daejeon, Korea) was used as DNA molecular weight marker. The
agarose gels electrophoresed were stained with ethidium bromide. The fragments
were illuminated with ultraviolet ray and then photographed by photoman direct
copy system (PECA products, Beloit, WI, USA).
3
DPPH Antioxidant and Antimicrobial Assays
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In this experiment, DPPH (1,1-diphenyl-2-picrylhydrazyl) (Sigma-Aldrich, USA), folin&ciocalteu’s reagent (sigma-Aldrich, USA), bacto agar (BD DIFCO, USA), bacto tryptic soy broth (BD DIFCO, USA), NO (nitric oxide) kit (Cell Biolabs. INC., USA), ELISA (enzyme-linked immunosorbent assay) kit (Abfrontier, KOR), TRIzol reagent (Sigma-Aldrich, USA), chloroform (Daejung, KOR), isopropyl alcohol (Sigma-Aldrich, USA), ethanol (Daejung, KOR), DEPC (diethyl pyrocarbonate) water (Thermo Scientific, USA), agarose (Bioneer, KOR), RT-PCR premix kit (Bioneer, KOR), Hematoxylin (Sigma-Aldrich, USA), Eosin Y (Sigma-Aldrich, USA ), centrifuge (Labogene, KOR), enzyme-linked immunosorbent assay (ELISA) reader (Thermo Scientific, USA), fluoro box (Neo Science, KOR), and Permount (Fisher, USA) were used.
4
Shrinkage and Conductivity of APPLE-Treated Agarose Gels
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agarose gel was prepared by dissolving 1% agarose (Bioneer) in boiling water followed by cooling down to room temperature.
The gel was cut to a fixed size of 0.5 cm in height and 1 cm in diameter for the shrinkage test. One of the gels was dip coated with APPLE solution using the method mentioned above, while the other gel was treated with distilled water as a negative control. The gels were then placed on top of a lidless membrane filter (Corning, 0.22 mm pore, cellulose acetate), the bottom of which was connected to a vacuum pump, and changes in the height of the gels were recorded over time. In addition, the decrease in conductance was monitored as the gels were dehydrated under vacuum. APPLE-treated and bare agarose gels were placed in an enclosed vacuum chamber (gauge pressure = À0.08 MPa) over 3 hours, and the resistivity of the gels was measured using a digital multimeter (Hontek, A830L) with the prongs touching the edges of the gels keeping them 1 cm apart from each other. Then, the conductance was calculated to be 1/the measured resistance. The surface morphology of the APPLE-treated gel was obtained using a field emission microscope (Magellan 400, FEI Company) with an accelerating voltage of 5 kV.
The gel was cut to a fixed size of 0.5 cm in height and 1 cm in diameter for the shrinkage test. One of the gels was dip coated with APPLE solution using the method mentioned above, while the other gel was treated with distilled water as a negative control. The gels were then placed on top of a lidless membrane filter (Corning, 0.22 mm pore, cellulose acetate), the bottom of which was connected to a vacuum pump, and changes in the height of the gels were recorded over time. In addition, the decrease in conductance was monitored as the gels were dehydrated under vacuum. APPLE-treated and bare agarose gels were placed in an enclosed vacuum chamber (gauge pressure = À0.08 MPa) over 3 hours, and the resistivity of the gels was measured using a digital multimeter (Hontek, A830L) with the prongs touching the edges of the gels keeping them 1 cm apart from each other. Then, the conductance was calculated to be 1/the measured resistance. The surface morphology of the APPLE-treated gel was obtained using a field emission microscope (Magellan 400, FEI Company) with an accelerating voltage of 5 kV.
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