Portions (25 μl) of each elution fraction from avidin-agarose affinity chromatography were boiled in 2x Laemmli buffer and loaded to a 10% SDS-PAGE gel. Electrophoresis was carried out at 200V for 60 minutes. After completion, the gel was washed in dH2O for 10 minutes. Then the gel was incubated with fixing solution (10% methanol, 7% acetic acid) for 1 hour at room temperature in an orbital shaker set at 50 rpm, followed by overnight incubation with SYPRO Ruby protein gel stain at room temperature with shaking and protection from light. The gel was then transferred to a clean staining container, and washed for 5 minutes with fixing solution followed by washing for 5 minutes in dH2O.Finally, the gel was imaged in a Molecular Imager Gel Doc system (Bio Rad, Hercules) using the highest sensitivity of the CCD camera (CoolSNAP HQ2, Roper Scientific) at a resolution of 1392 x 1040 pixels with 12 bit gray scale levels per pixel.
Molecular imager gel doc system
Manufactured by Bio-Rad
Sourced in United States
The Molecular Imager Gel Doc system is a laboratory instrument designed for imaging and analysis of gels, blots, and other gel-based samples. It captures high-quality digital images for documentation and quantitative analysis.
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Lab products found in correlation
Coolsnap hq2, by Teledyne (1 mentions)
Dna ladder, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Image lab software v 6, by Bio-Rad (1 mentions)
Coomassie blue r 250, by Merck Group (1 mentions)
Rna loading dye, by New England Biolabs (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
5 protocols using molecular imager gel doc system
1
SYPRO Ruby Protein Gel Staining
2
Agarose Gel Electrophoresis Analysis of PCR Products
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PCR products were analyzed using agarose gel electrophoresis (1.5%) and visualized with ethidium bromide (10 μg/μl) staining. The gels were exposed to UV light and photographed using a Molecular Imager® Gel Doc™ System with Image Lab™ Software, Bio-Rad. The size of the DNA fragments was estimated based on a DNA ladder (100 to 2000 bp, MBI, Fermentas). The presence or absence of each band was treated as binary character in a data matrix, that is, coded 1 and 0, respectively, The amplification products were scored as (1) for the presence and (0) for the absence of the bands and were compared to the bands in the control group to determine the genetic alterations across the other treated groups. The appearance of new bands and disappearance of existed bands in comparison with the control group are considered DNA polymorphism. Percentage of polymorphism is calculated according to this equation: number of polymorphic DNA band × 100/total number of bands.
3
Agarose Gel Electrophoresis of DNA
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Gel electrophoresis analyses were performed using either 1 or 2% (w/v) agarose dissolved in 1× TAE buffer (39.95 mM Tris-base, 11.42% (v/v) glacial acetic acid, 127.29 μM EDTA) [23 ]. One or two microliters of 6× DNA loading dye (Fermentas Life Sciences, MA, USA) was mixed with 10 µL of DNA or 1 µL of GeneRuler™ 1 Kbp or 100 bp DNA ladder (Fermentas Life Sciences, MA, USA) for loading into the gels and subsequent electrophoresis and image capture (Molecular Imager Gel Doc System, Bio-Rad Laboratories, CA, USA).
4
SDS-PAGE Protein and Carbohydrate Visualization
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Proteins and carbohydrates were visualized through the use of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described [41 (link)]. Gels containing 15% bis-acrylamide were loaded with 40 µL of protein samples suspended in a loading buffer with final concentration of 0.0175 M Tris-HCl (pH 6.8), 0.8% SDS, 9% glycerol, 2.5% β-mercaptoethanol and 0.002% bromophenol blue, and run on a Bio-Rad Mini-Protean® Tetra Cell System (Bio-Rad Laboratories, Hercules, CA, USA). Electrode chambers were filled with Tris-Glycine buffer (50 mM Tris, 200 mM glycine, 0.2% SDS). For protein visualization, gels were stained overnight in staining solution (1 g Coomassie blue R250 (Merck, Darmstadt, Germany) in 50% (v/v) ethanol, 10% (v/v) acetic acid), and destained with 12.5% isopropanol and 10% (v/v) acetic acid. For carbohydrate visualization, gels were stained using the periodic acid-Schiff (PAS) protocol described by Kapitany and Zebrowski [42 (link)]. Images of the gels were captured using a Molecular Imager® Gel Doc™ System (Bio-Rad Laboratories) using Image Lab™ Software v6.0 (Bio-Rad Laboratories).
5
Cas13a Cleavage Kinetics at Varying Temperatures
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HheCas13a and TccCas13a cleavage reactions were performed at 37°C and 60°C with synthetic, in vitrotranscribed RNA targets. Brie y, for both HheCas13a or TccCas13a cleavage assays, cleavage reactions were carried out in 20-mL reaction volume with 50 nM of either Cas13a protein, 50 nM of their cognate crRNAs, and 100 nM of target RNA in 1x isothermal buffer (20 mM Tris-HCl pH 8.8, 50 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Tween 20) supplemented with an additional 6 mM MgSO 4 ( nal of 8 mM MgSO 4 ); the reactions were then incubated at the indicated temperatures for 1 h (no pre-assembly of Cas13a protein and crRNA to form RNP was performed). The samples were then boiled at 70°C for 3 min in 2X RNA Loading Dye (B0363S, NEB) and cooled down on ice for 3 min before loading onto a 6% polyacrylamide-urea denaturing gel. Electrophoresis was conducted for 45 min at 25 W. The gel was stained with SYBR Gold Nucleic Acid Gel Stain (S11494, ThermoFisher) for 10 min, brie y washed with 1X Tris borate EDTA buffer and visualized using a Bio-Rad Molecular Imager Gel Doc system.
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