PAGE-purified XNAzymes and RNA substrates were annealed separately as described above and reacted in ‘quasi-physiological buffer’ (30 mM EPPS pH 7.4, 150 mM KCl, 1 mM MgCl2) at 37 °C. Reactions were stopped by addition of PAGE gel loading buffer (95% formamide, 20 mM Tris pH 7.5, 10 mM EDTA, 0.05% bromophenol blue) and analysed by Urea-PAGE using 20% acrylamide gels. Gels containing fluorophore-labelled RNA substrates and 5’ RNA products were imaged without staining using an FLA-5000 scanner (Fujifilm, Japan); unlabelled RNA substrates and products were first stained using SYBR Gold stain (Thermo Fisher Scientific, USA). Bands were quantified using Fiji102 (link) to calculate proportion of RNA cleaved for each XNAzyme reaction. For SYBR Gold stained RNA products, a coefficient to account for differential staining proportional to oligo length was applied to product band densities. Pseudo first-order reaction rates (kobs) under single-turnover pre-steady-state (Km/kcat) conditions were determined from time courses; samples were taken and reactions stopped at appropriate intervals by snap-freezing on dry ice in excess PAGE gel loading buffer. Quantification data from three independent replicates per time course were fit using Prism 9 (GraphPad), as described previously62 (link).
Sybr gold stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYBR Gold stain is a fluorescent dye used for detecting and quantifying nucleic acids, including DNA and RNA, in various applications such as gel electrophoresis and real-time PCR. It binds to the nucleic acids, allowing them to be visualized under ultraviolet (UV) or blue light.
Automatically generated - may contain errors
Lab products found in correlation
Bis tris native page, by Thermo Fisher Scientific (3 mentions)
Multigauge, by GE Healthcare (3 mentions)
Maxiscript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Tbe polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Gel doc ez imager, by Bio-Rad (2 mentions)
Ultrapure agarose, by Thermo Fisher Scientific (2 mentions)
Blue transilluminator, by Thermo Fisher Scientific (2 mentions)
Ficoll based loading solution, by Promega (2 mentions)
Tbe gel, by Thermo Fisher Scientific (2 mentions)
Fla 9000, by Fujifilm (2 mentions)
Prism 9, by GraphPad (1 mentions)
Fla 5000 scanner, by Fujifilm (1 mentions)
T4 polynucleotide kinase pnk, by New England Biolabs (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Xrf gel doc system, by Bio-Rad (1 mentions)
Loading buffer, by Takara Bio (1 mentions)
20 bp dna ladder dye plus, by Takara Bio (1 mentions)
T100 thermocycler, by Bio-Rad (1 mentions)
Human thrombin, by Merck Group (1 mentions)
Firepol master mix, by Solis BioDyne (1 mentions)
41 protocols using sybr gold stain
1
Characterizing XNAzyme Kinetics
2
Csm RNA and DNA Cleavage Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TthCsm-TEC pull-down elution (2 µl) was diluted to 10 µl with HN100 buffer. Reactions were initiated by adding 5 mM MgCl2 to allow RNA cleavage or 5 mM MnCl2 to allow both ssDNA and RNA cleavage. Following incubation at 65 ˚C for 30 min, reactions were quenched with 1 volume of 2X Gel Loading Buffer II (Invitrogen). Reactions were analyzed by denaturing 15% urea PAGE in 0.5× TBE buffer and SYBR gold stain (Thermo Fisher) was used to visualize ssDNA and ssRNA.
For radioactive cleavage assays with the TEC, assembly of the TEC and pull-downs with TthCsm were performed as described above, except that a trace amount of nontemplate DNA was 5´-end-labeled with [γ-32P]-ATP (Perkin-Elmer) using T4 polynucleotide kinase (PNK; New England Biolabs) and incorporated into the TEC. For reactions with free nontemplate strand substrate, 0.8 µM of nontemplate DNA (Supplementary Table 1, TthNTS1 or TthNTS2) was mixed with 0.2 µM TthCsm and 0.2 µM of the target RNA transcript. The same was done for reactions using the empty R-loop, except 0.8 µM of nontemplate DNA was annealed with an equimolar amount of the template strand (Supplementary Table 1, TthTS1 or TthTS2), forming a DNA mismatch bubble. Cleavage reactions (20 µl) were initiated by addition of 5 mM MnCl2. A 5´-end-labeled ssDNA ladder was generated using truncations of the nontemplate strand DNA.
For radioactive cleavage assays with the TEC, assembly of the TEC and pull-downs with TthCsm were performed as described above, except that a trace amount of nontemplate DNA was 5´-end-labeled with [γ-32P]-ATP (Perkin-Elmer) using T4 polynucleotide kinase (PNK; New England Biolabs) and incorporated into the TEC. For reactions with free nontemplate strand substrate, 0.8 µM of nontemplate DNA (Supplementary Table 1, TthNTS1 or TthNTS2) was mixed with 0.2 µM TthCsm and 0.2 µM of the target RNA transcript. The same was done for reactions using the empty R-loop, except 0.8 µM of nontemplate DNA was annealed with an equimolar amount of the template strand (Supplementary Table 1, TthTS1 or TthTS2), forming a DNA mismatch bubble. Cleavage reactions (20 µl) were initiated by addition of 5 mM MnCl2. A 5´-end-labeled ssDNA ladder was generated using truncations of the nontemplate strand DNA.
3
T7 RNA Polymerase Stop Assay for G4 Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The T7 RNA polymerase stop assay was performed using the protocol based on the study by Tateishi-Karimata et al. (20 (link)). Oligonucleotides (Supplementary Table S2 ) containing the T7 polymerase binding site and a 35nt-spacer followed by the G4 forming sequence were synthesized and annealed with the complementary strand to form a dsDNA T7 polymerase binding site while leaving the spacer and G4-forming site single-stranded. The list of oligonucleotides used for this study is listed in Supplementary Table S2 . The reaction was performed by incubating T7 polymerase and the annealed oligonucleotides for 10 mins in 1× T7 Polymerase Reaction Buffer (NEB), followed by addition of rNTPs and further incubation at 37°C for 90 mins. The reaction was quenched using Stop Buffer (80% formamide, 10mM MgCl2 and 0.01% Dextran Blue). The samples were heated for 5 min at 95°C before being loaded on a 10% denaturing PAGE gel with 7M urea and electrophoresed for 45 min at 60°C and stained with SYBR Gold stain (Thermo Scientific). The gels were imaged on the Bio-Rad XRF Gel Doc system.
4
Temperature-Dependent Folding of cspA 5' UTR
5
Temperature-Dependent Folding of cspA 5' UTR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Matrices for in vitro transcription were amplified from the plasmids pBAD2-bgaB containing cspA 5′ UTR and its mutated forms with primers cspA_T7_F and cspA_T7_R (Table S6 ). In vitro transcription was performed with MAXIscript T7 Transcription Kit (Thermo Fisher Scientific) and the synthesized RNA was purified on 6% denaturing polyacrylamide gel (19:1 acrylamide/bis-acrylamide ratio). To study the effect of temperature on folding, 50 ng of cspA 5′ UTR was dissolved in 9 μL of Loading buffer (10 mM Tris-HCl pH7.0, 1 mM EDTA, 10% glycerol, 0.01% xylene cyanol), denatured at 95°C for 3 minutes and quickly cooled on ice. To initiate refolding, 1 μl of 500 mM NaCl solution was added, and RNA was incubated at temperatures ranging from 26°C to 37°C for 5 minutes. The folded RNA was applied to the 10% acrylamide gel containing 100 mM Tris-HEPES (pH = 7.5), 0.1 mM EDTA, 3 mM MgCl2, and run at 4°C with a running buffer of the same composition. The RNA fragments in the gel were visualized by staining with SYBR Gold stain (Thermo Fisher Scientific).
6
Acrylamide Gel Electrophoresis for DNA Hybridization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The intended DNA hybridization was confirmed by gel electrophoresis. First, input DNA and complementary DNA were mixed with 10× loading buffer (Takara Bio Inc., Shiga, Japan). Then, 15% acrylamide gel (ATTO Corp., Tokyo, Japan) was prepared using Tris-borate-EDTA buffer (0.089 M Tris boric acid and 2.6 mM EDTA, pH 8.3–8.5; Takara Bio Inc.). The DNA sample was electrophoresed at 150 V for 90 min along with a reference marker [20-bp DNA Ladder (Dye Plus); Takara Bio Inc.]. The gel was visualized by staining in SYBR gold stain (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 15 min. The resultant gel revealed a main band of dsDNA at the same location as the 40-bp reference dsDNA (S1 Fig ), inferring that DNA hybridization between input DNA and complementary DNA had occurred as designed.
7
Validation of Mitochondrial and Linear DNA Removal
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate removal of mitochondrial DNA and linear DNA, 50 ng each of crude eccDNA and eccDNA purified, with the same amount of gDNA as a positive control, was used to amplify targeted regions of the mouse gene Actb and Cox5b and the mouse mitochondrial gene mt-Co1 [41 (link), 45 (link)]. PCR products were resolved by DNA gel electrophoresis and stained by either ethidium bromide or SYBR Gold stain (Thermo Fisher, Catalog: #S11494) for higher sensitivity [47 (link)]. The primer pairs used for PCR included the following: 5′-GAGACCTTCAACACCCCAG-3′ (forward) and 5′-TCAGGGCATCGGAACCG-3′ (reverse) for a 404-bp region of Actb, 5′-GCCCATTTCCACTATGTTCTA-3′ (forward) and 5′-AGTAGCCTGCTCCTCATCAG-3′ (reverse) for a 119-bp region of Cox5b, and 5′-GCCCATTTCCACTATGTTCTA-3′ (forward) and 5′-GTTTACTCCTACGAATATGATG-3′ (reverse) for a 144-bp region of mt-Co1.
8
Oligonucleotide Library Formation and Thrombin Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Both the forward and reverse primers of sequences, 5′ATC AGT TCG AGC AGA TGA GC′3 and 5′CCA GAC TGC GAG CGT TTT TTT TTT-3′ respectively as well as a 10–100 nt oligonucleotide ladder, were purchased from IDT. Candidate polynucleotide sequences were also purchased from IDT using their Ultramer® technology including two scrambled sequences (SC01 and SC02 ), which correspond to the randomised sequence of the longest polynucleotide sequences tested. Terminal deoxynucleotidyl transferase (TdT), dNTPs, and human thrombin were purchased from Sigma Aldrich. SYBR gold stain was purchased from Thermo Scientific. 5x FIREPol® Master Mix and sample loading buffer were purchased from Solis Biodyne. The Buffer SB1: 50 mM Tris–HCl, 250 mM KCl and 7.5 mM MgCl2 pH 7.4 (5×) were used for all TdT catalysed library formation reactions and library selection steps. All oligonucleotide purification steps were performed using an oligonucleotide purification kit from Norgen Biotek. Purification of the PCR products was performed using a PCR clean-up kit purchased from Macherey-Nagel GmbH. All electrophoretic mobility shift assays (EMSA); (5–6%) and agarose gels (2%) were prepared in house. All PCR reactions were prepared in a dedicated laminar flow cabinet and all PCR experiments were performed on a T100 thermocycler (BioRad).
9
Genomic DNA Isolation and PCR Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three days post transduction, genomic DNA was isolated using QuickExtract (catalog #QE09050, Epicenter) according to the manufacturer’s instructions. PCR amplification of the genomic DNA was performed using 700 ng genomic DNA and specifically designed forward and reverse primers (Supplementary Table 3 ) and KAPA DNA polymerase (KAPA Biosystems). The PCR product was purified and 315 ng of the purified DNA was denatured and reannealed in the presence of NEB restriction enzyme buffer 2 in a thermocycler. The reannealed DNA was mixed with enhancer and Surveyor nuclease (IDT) according to the manufacturer’s instructions and incubated at 42 °C for 50 min before separation on 4–20% Tris/Borate/EDTA gels. The DNA bands were visualized using SYBR gold stain (Thermo Scientific).
10
MORC2 Binding to Nucleosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of 10 μl containing 100 ng of 601 mononucleosome positioning sequence DNA (a kind gift from T. Bartke, Helmholtz Zentrum, Munich) or 500 nM NCP (a kind gift from S. Tan, Penn State University) were incubated on ice with increasing concentrations of MORC2 variants in gel filtration buffer for 1 h. The reaction mixtures were loaded on a 6% polyacrylamide gel that had been prerun on ice for 1 h at 150 V in 45 mM (0.5×) Tris-borate buffer. Electrophoresis was performed at 150 V on ice for 90 min. The gels were post-stained for DNA with 1× SYBR gold stain (Thermo Fisher Scientific), then visualized with the G-BOX system (Syngene), or with 2 µM SYTO 62 (Invitrogen), and then visualized with the Odyssey CLx system (LICOR).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!