Nanoswitches were run in 0.8% agarose gels, cast from molecular biology grade agarose (Fisher BioReagents) dissolved in 0.53× Tris-borate EDTA (TBE). Gels were typically run at 75 V (constant voltage) at room temperature. Samples were prestained by mixing 1× GelRed stain with the samples before loading. Gels were imaged with a Bio-Rad Gel Doc XR+ gel imager and analyzed using ImageJ.
Molecular biology grade agarose
Manufactured by Thermo Fisher Scientific
Sourced in United States
Molecular biology grade agarose is a purified polysaccharide used as a gel matrix for the separation and analysis of nucleic acids (DNA and RNA) in electrophoresis applications. It serves as a support medium for the migration of charged molecules under the influence of an electric field.
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Lab products found in correlation
Gel doc xr gel imager, by Bio-Rad (2 mentions)
Gel doc xr, by Bio-Rad (2 mentions)
Gelred nucleic acid stain, by Biotium (2 mentions)
M13 circular dna, by New England Biolabs (2 mentions)
Btsci enzyme, by New England Biolabs (2 mentions)
Rnase h, by New England Biolabs (2 mentions)
Oligonucleotide, by Integrated DNA Technologies (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Typhoon 9400 variable mode imager, by GE Healthcare (1 mentions)
Gelred stain, by Biotium (1 mentions)
Direct q 5, by Merck Group (1 mentions)
Cell counting kit 8 cck 8 assay, by Dojindo Laboratories (1 mentions)
Huvecs, by National Collection of Authenticated Cell Cultures (1 mentions)
Folate, by Merck Group (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindotricarbocyanine iodide dir, by Thermo Fisher Scientific (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate dii, by Merck Group (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Human umbilical vein endothelial cells huvecs, by National Collection of Authenticated Cell Cultures (1 mentions)
A549 human lung adenocarcinoma cells, by National Collection of Authenticated Cell Cultures (1 mentions)
9 protocols using molecular biology grade agarose
1
Agarose Gel Electrophoresis of Nanoswitches
2
Gel Electrophoresis Protocol for Nucleic Acid Analysis
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Constructs were run in 0.8% agarose gels, cast from molecular biology grade agarose (Fisher BioReagents) dissolved in 0.5× Tris-borate EDTA (TBE) (Ultra pure grade, Amresco). Samples were mixed with a Ficoll-based loading solution. Gels were typically run at 75 V (constant voltage) at room temperature. Gels were pre-stained by mixing 1× GelRed stain (Biotium) with the gel solution before the gel was cast. Gels were imaged with a Bio-Rad Gel Doc XR+ gel imager and analyzed using the gel analysis tool in the Image Lab software package available with Bio-Rad Gel Doc XR+. For the Hello World → Good Bye multi-bit rewriting experiment, gels were run at 100 V (constant voltage) and imaged using a Typhoon 9400 variable mode imager (GE Healthcare). Image analysis was done using the software ImageJ (https://imagej.nih.gov/ij/ ). Median filter in ImageJ was used to remove noise in gel images in Figures 2D , 3F and 4A –B .
3
Gel-based Detection Efficiency Analysis
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The detection samples were run in 25-ml 0.8% agarose gels unless otherwise noted, cast from molecular biology grade agarose (Fisher BioReagents) dissolved in 0.5× TBE buffer. Typical running conditions were 75 V for 45 to 70 min at room temperature or cold room. Samples were mixed with a Ficoll-based blue loading dye before loading. Imaging was completed on a Bio-Rad Gel Doc XR+ imager with different exposure times based on the brightness of the detection bands. The detection efficiency was analyzed using included Image Lab software (Fig. 2E ). The profiles of detection bands were obtained in ImageJ (57 (link)), and then their integrated intensities were obtained by using the peak analysis function in Origin (OriginLab Corporation), such as the data presented in Figs. 2F , 4C , and 5 (A and B) . The detailed analysis procedure can be found in our previous publication (21 (link)). For the E-Gel–related experiments, we used Invitrogen E-Gel agarose system (Thermo Fisher Scientific) and its precast agarose gel (1.0%, SYBR stained). Ten microliters of nanoswitch detection sample was loaded to each lane, and the gel was run at 48 V for 1 hour at room temperature. Because the E-Gel system does not allow user control of the voltage, we used an external power supply connected with the negative and positive electrodes of the precast agarose gel to supply 48 V.
4
M13mp18 DNA Nanoswitches Construction
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Oligonucleotides were purchased from Integrated DNA Technologies (IDT) with standard desalting. M13 circular DNA, RNase H, RNase T (Exo T), RNase If, and BtsCI enzymes were purchased from New England Biolabs (NEB). GelRed nucleic acid stain was purchased from Biotium. Molecular biology grade agarose was purchased from Fisher BioReagents. We have used the viral genome M13mp18 (7,249 nt) for this and previous constructions of our nanoswitches due to its commercial availability and frequent use in DNA origami.
5
M13mp18 DNA Nanoswitches Construction
6
Agarose Gel Electrophoresis of Nanoswitches
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Nanoswitches were run in 0.8% agarose gels, cast from molecular biology grade agarose (Fisher BioReagents) dissolved in 0.53× Tris-borate EDTA (TBE). Gels were typically run at 75 V (constant voltage) at room temperature. Samples were prestained by mixing 1× GelRed stain with the samples before loading. Gels were imaged with a Bio-Rad Gel Doc XR+ gel imager and analyzed using ImageJ.
7
ZIKV RNA Detection Assay with Nanoswitches
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For the experiment in Fig. 2C , 5 ng (~8.5 × 108 copies) of ZIKV RNA was used in 10 μl of detection assay. Samples were run in 25 ml of 0.8% agarose gels, cast from molecular biology grade agarose (Fisher BioReagents) dissolved in 0.5× tris-borate-EDTA (TBE) buffer. For the experiments in Fig. 2F and fig. S8, first, all nanoswitches were purified by LC and then their concentrations were determined by measuring A260 absorbance with a Thermo Fisher Scientific NanoDrop 2000. Nanoswitch mixtures were made by mixing nanoswitches in equimolar concentrations. The detection reaction volume is 10 μl with nanoswitch (100 pM final concentration), MgCl2 (10 mM), 1× PBS, and blocking oligos (200 nM). The blocking oligos are short oligos (14 nucleotides) that can prevent the binding of target RNA to the inner surface of plastic tubes (21 (link)). Samples were incubated in a thermal cycler with thermal annealing from 40° to 25°C over ~12 hours (at −0.1°C per cycle and 5 min for each cycle, for a total of 150 cycles).
8
Multifunctional Nanoparticle-based Drug Delivery
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1,2-Distearoyl-sn-glyc-ero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-2000] (DSPE-PEG (2000)-folate), cholesterol, PFP, ICG, DOX, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), Hoechst 33342, were brought from Sigma-Aldrich Co. (St. Louis, MO). 1,2-Dihexadecanoyl-rac-glycero-3-phoshocoline (DPPC) was purchased from Xi’an Ruixi Biological Technology (Xi’an, China). Chloroform (CHCl3) was purchased from Chongqing East Chemical Industry Ltd., Co. (Chongqing, China). Molecular biology-grade agarose was purchased from Thermo Fisher Scientific (Waltham, MA). Monoclonal antibodies against folic acid in mice (primary antibody) were obtained from Beijing Hapten and Protein Biomedical Institute (Beijing, China). FITC-labeled anti-mouse IgG antibodies in sheep (second antibody) were purchased from Abcam (Waltham, MA). The human RB cell line Y79 and human umbilical vein endothelial cells (HUVECs) were purchased from the China Center for Type Culture Collection (Wuhan, China). The cell counting kit-8 (CCK-8) assay was obtained from Dojindo Laboratories (Kumamoto, Japan). Deionized water obtained from the Millipore system (Direct-Q5, FRA, Billerica, MA) was used in all preparations.
9
Nanoparticle-mediated Targeted Drug Delivery
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ICG, polyvinyl alcohol, folate, Ptx, DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), DAPI (4′,6-diamidino-2-phenylindole) were purchased from Sigma-Aldrich (St Louis, MO, USA). Methoxy-PEG-PLGA (molecular weight [MW] 12,000 Da), carboxyl-PLGA (COOH–PLGA) (MW 12,000 Da), and amino-PEG-amino (NH2-PEG-NH2) were purchased from Daigang Biomaterial (Jinan, China). Molecular biology-grade agarose and DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Pfh was purchased from Apollo Scientific (Stockport, UK). MDA-MB231 human breast cancer cells, A549 human lung adenocarcinoma cells, and human umbilical vein endothelial cells (HUVECs) were purchased from the China Center for Type Culture Collection (Wuhan, China). Deionized water obtained from the Millipore system (Direct-Q 5, FRA) was used in all preparations.
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