ExoG (1.25, 25 or 50 nM) was incubated with FAM (6-carboxyfluorescein)-labeled substrates (100 nM) in a 10 μL reaction containing 100 μg mL−1 bovine serum albumin (BSA), 10 mM HEPES pH 7.4, 150 mM NaCl and 2.5 mM MgCl2 at 37°C. For biotin-labeled substrates, an additional 200 nM NeutrAvidin (Thermo Scientific) was included in the reaction. Oligonucleotide sequences are listed in Supplementary Table S1 . Reactions were stopped at the indicated time-points by adding an equal amount of 2X TBE/urea sample buffer (BIO-RAD) and heating at 65°C for 20 min. To fully release the FAM-labeled probe from the complementary strand, 2 μL of 100 μM competitive DNA oligonucleotides was added. The resultant mixtures were heated at 95°C for 5 min, then at 30°C for 10 min, before being gradually cooled to room temperature (∼20°C). The solutions were loaded and separated by a 20% denatured acrylamide gel containing 6 M urea. FAM-labeled oligonucleotides (excitation at 473 nM and emission at 520 nM) in the resultant gels were visualized using a Typhoon FLA 9000 biomolecular imager (GE Healthcare Life Sciences). Quantification of band signal was plotted in GraphPad Prism v. 7.0 (34 ).
Tbe urea sample buffer
Manufactured by Bio-Rad
TBE-urea sample buffer is a laboratory reagent used in DNA or RNA electrophoresis applications. It is designed to prepare samples for separation and analysis on polyacrylamide or agarose gels.
Automatically generated - may contain errors
Lab products found in correlation
Gelred, by Biotium (2 mentions)
Typhoon fla 9000, by GE Healthcare (1 mentions)
Neutravidin, by Thermo Fisher Scientific (1 mentions)
Typhoon 9400, by GE Healthcare (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Dig wash and block buffer set, by Roche (1 mentions)
Small rna marker, by Abnova (1 mentions)
Ultra hyb oligo hybridisation buffer, by Thermo Fisher Scientific (1 mentions)
Dig luminescent detection kit, by Roche (1 mentions)
Mini trans blot electrophoretic transfer cell, by Bio-Rad (1 mentions)
Protean 2 xi cell, by Bio-Rad (1 mentions)
Acrylamide and bis acrylamide solution, by Bio-Rad (1 mentions)
Tbe buffer, by Bio-Rad (1 mentions)
Rnase r, by Illumina (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Mini protean tetra cell, by Bio-Rad (1 mentions)
Decade marker, by Thermo Fisher Scientific (1 mentions)
Tbe urea gel, by Thermo Fisher Scientific (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
9 protocols using tbe urea sample buffer
1
Quantitative Exonuclease G Assay
2
Urea-PAGE Analysis of Primer Extensions
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Completed extensions were analyzed by urea-PAGE. 8 μL of each completed primer extension reaction was combined with 12 μL of BioRad 2x TBE urea sample buffer and boiled for 10 minutes.
The boiled samples were loaded onto a 10% polyacrylamide TBE urea gel (bioRad 4566036), and 200V was applied to the gel for 40 minutes. The gels were imaged on a GE Healthcare Typhoon 9400 laser scanner using a 200um pixel size and λex=488nm, λem=520nm BP40. Imaging gain was adjusted for each experiment to avoid saturation.
The boiled samples were loaded onto a 10% polyacrylamide TBE urea gel (bioRad 4566036), and 200V was applied to the gel for 40 minutes. The gels were imaged on a GE Healthcare Typhoon 9400 laser scanner using a 200um pixel size and λex=488nm, λem=520nm BP40. Imaging gain was adjusted for each experiment to avoid saturation.
3
RNA Substrate Processing Assay
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The processing assay was carried out at 37°C in 20 μl assay buffer containing 50 mM Tris-hydrochloride (Tris–HCl, pH 7.5), 150 mM sodium chloride (NaCl), 10% glycerol, 0.2 μg/μl bovine serum albumin (BSA), 1 mM dithiothreitol (DTT), and 2 mM magnesium chloride (MgCl2). Approximately 10 000 cpm of the RNA substrates was used, and the enzyme concentrations and incubation time are indicated in the figures. The reaction was stopped by adding 20 μl TBE-urea sample buffer (Bio-Rad) and immediately chilling the mixture on ice. Finally, the mixture was heated at 95°C for 10 min and quickly chilled on ice before being loaded onto a 10% Urea-polyacrylamide gel electrophoresis (Urea-PAGE) gel with the RNA size markers (Decade marker, Ambion). The RNA substrates were prepared by the in vitro transcription using MEGAscript T7 Kit (Ambion). The substrates were either internally labeled with [α-32P] UTP or end-labeled with [γ-32P] UTP.
4
Dual Incision Assay for Nucleotide Excision Repair
5
Northern Blot Analysis of RNA Samples
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Pre-cast 10% polyacrylamide TBE-urea gels (Bio-Rad) were loaded with 5 µg total RNA per lane. RNA samples were mixed with TBE-urea sample buffer (Bio-Rad) and denatured at 90 °C for 5 min. On each gel, two RNA ladders were run alongside: 0.1–2 kb RNA ladder (Invitrogen) and a small RNA marker (Abnova, fragment sizes 20 to 100 bases). Gels were run for 1 to 1.5 hours at 80 V. Before blotting, lanes containing RNA ladders were cut off the gel, stained with GelRed (Biotium), and the band position documented for later RNA size estimation.
RNA was blotted onto positively charged nylon membranes (Roche) in a Mini Trans-Blot Electrophoretic Transfer cell (Bio-Rad) for 1 hour at 300 mA, then cross-linked with UV-light. 5′DIG-labelled probes were obtained from Sigma. Membranes were hybridised at 42 °C overnight at 8 rpm, with 100 ng/ml DIG-labelled probes (TableS6 ) in ULTRA-Hyb-Oligo hybridisation buffer (Ambion). Membrane washing and immunological detection of probes was performed using the DIG Wash and Block Buffer Set and the DIG Luminescent Detection Kit (Roche). Luminescence was recorded with BioMax Light X-ray films (Carestream). Membranes were stripped using 0.5% SDS at 60 °C for one hour and re-used for hybridisation. Northern blots were conducted in triplicate, using RNA isolated from three independent biological replicates.
RNA was blotted onto positively charged nylon membranes (Roche) in a Mini Trans-Blot Electrophoretic Transfer cell (Bio-Rad) for 1 hour at 300 mA, then cross-linked with UV-light. 5′DIG-labelled probes were obtained from Sigma. Membranes were hybridised at 42 °C overnight at 8 rpm, with 100 ng/ml DIG-labelled probes (Table
6
Purification of RNA Topological Structures
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Gels of different concentrations were prepared using 30% acrylamide and bis-acrylamide solution (Bio-Rad, 29:1) with 7 M urea in 0.5 × TBE buffer (Bio-Rad) and run on a PROTEAN II xi cell (Bio-Rad) or a Mini-PROTEAN Tetra cell (Bio-Rad). Samples were mixed 1:1 with TBE-urea sample buffer (Bio-Rad) and heated at 70 °C for 5 min before they were loaded into the wells. Gel concentrations were carefully chosen to ensure the proper separations between different topologies as well as the references. For imaging, gels were stained with GelRed (Biotium), and images were taken by Gel Doc XR+ (Bio-Rad) imaging system and processed by software Image Lab (v.4.0.1, Bio-Rad). For purification, gels (without staining) were visualized by UV shadowing against a fluorescent thin-layer chromatography plate and bands of interest were cut. The bands were then eluted using the crush-and-soak method and the eluent was purified and concentrated on 3 K Nanosep filters (Pall). The concentration of product was determined by measuring the OD260. Optionally, the ssRNA knots and circles can be digested by RNase R (Epicentre) after the gel extraction to remove the unavoidable cleaved linear RNA during the purification. However, RNase R digestion is not useful for the hybrid BR.
7
RNA Substrate Processing Assay Protocol
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The processing assay was carried out at 37°C in 20 μL assay buffer containing 50 mM Tris-hydrochloride (Tris-HCl [pH 7.5]), 100 mM sodium chloride (NaCl), 10% glycerol, 0.2 μg/μL bovine serum albumin (BSA), 1 mM dithiothreitol (DTT), and 2 mM magnesium chloride (MgCl2). Approximately 10,000 cpm of the RNA substrates was used, and the enzyme concentrations and incubation time are indicated in the figures. The reaction was stopped by adding 20 μL TBE-urea sample buffer (Bio-Rad). Finally, the mixture was heated at 95°C for 10 min and quickly chilled on ice before loading onto a 10% Urea-polyacrylamide gel electrophoresis (Urea-PAGE) gel with the RNA size markers (Decade marker, Ambion). The RNA substrates were prepared as described previously (Nguyen et al. 2015 (link)).
8
Polymerase Kinetic Assay for mtDNA
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DNA polymerization and 3′-5′ exonuclease activity were assayed using the same primer-template as described above for electrophoresis mobility shift assay (EMSA). The reaction mixture contained 10 fmol of the DNA template, 25 mM Tris-HCl [pH 7.8], 10% glycerol, 1 mM DTT, 10 mM MgCl2, 100 μg/ml BSA, 60 fmol of POLγA, 120 fmol of POLγB and the indicated concentrations of the four dNTPs. The reaction was incubated at 37 °C for 15 min and stopped by the addition of 10 μl of TBE-UREA-sample buffer (BioRad). The samples were analysed on a 15% denaturing polyacrylamide gel in 1 × TBE buffer.
9
Single-Stranded DNA Cleavage Assay
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Single-stranded DNA activity assays were performed at equimolar amounts of pAgo protein, guide and target DNA in 1x CutSmart buffer (New England Biolabs). The final concentration of protein, guide and DNA template in a total reaction volume of 15 µl is 250 nM. Proteins were prediluted to a concentration of 750 nM in 1x CutSmart buffer (New England Biolabs) and preloaded with guide DNA or guide RNA in a 1:1 ratio at 37°C for 15 min. The, single-stranded DNA template is added and incubated at 37°C or as indicated for 1h. To inactivate ssDNA cleavage reactions, samples were incubated with TBE urea sample buffer (Biorad) in a 1:1 ratio at 95°C for 10 min. ssDNA cleavage products were resolved on 15% TBE Urea gels (Invitrogen). As a marker, an in-house prepared ssDNA marker consisting of 21, 30, 60 and 90 nt oligos was used. Gels were stained for 10 min with SYBR gold Nucleic Acid Gel Stain (Invitrogen) and visualized using a UVsolo TS Imaging System (Analytik Jena). Sequence of the ssDNA templates T1 and T2 is shown in Supplementary Table 3. Sequence of the guide DNA and guide RNA is shown in Supplementary Table 4.
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