Nonspecific cleavage activity of Cpf1 was activated by incubating Cpf1, crRNA, and DNA activator with a concentration of 100 nM:100 nM: 2 nM in 1× NEBuffer 2.1 buffer at 37 °C for 30 min. M13mp18 was then added to the 30 μl reaction mixture and incubated for an additional 45 min. A fraction of the reaction was taken out every 5 min, quenched in 6× purple gel loading dye (New England Biolabs Inc.), and subsequently analyzed in 1% agarose gel (Fisher Scientific)1 (link).
Purple gel loading dye
Manufactured by New England Biolabs
Sourced in United States
Purple Gel Loading Dye is a ready-to-use solution used to prepare DNA samples for gel electrophoresis. It contains a purple tracking dye that allows visualization of the sample during loading and running of the gel.
Automatically generated - may contain errors
Lab products found in correlation
Agarose gel, by Thermo Fisher Scientific (1 mentions)
1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Ethidium bromide, by Carl Roth (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Quick cip, by New England Biolabs (1 mentions)
Q5 high fidelity pcr kit, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
M13mp18 circular ssdna, by New England Biolabs (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Agarose, by Merck Group (1 mentions)
1 kb plus dna ladder, by New England Biolabs (1 mentions)
Gelred, by Biotium (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
Proteinase k, by New England Biolabs (1 mentions)
Electrophoresis equipment, by Bio-Rad (1 mentions)
Quick load 1 kb extend dna ladder, by New England Biolabs (1 mentions)
16 protocols using purple gel loading dye
1
Cpf1 Nonspecific Cleavage Activation
2
Analyzing AAV Vector Genome Integrity
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To analyze the genome integrity of scAAV vectors, 5 × 1011 AAV particles (vector genomes as quantified by qPCR) were treated with Proteinase K (QIAGEN) for 30 min at 56°C. Next, vector DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN), mixed with 6× purple gel loading dye (NEB), and loaded next to the 1 Kb Plus DNA Ladder (Thermo Fisher) onto a native 1% agarose gel stained with ethidium bromide (Roth, Karlsruhe, Germany). Electrophoresis was performed in 1× TAE (tris-acetate-EDTA) buffer at 120 V for 30 min.
3
Efficient DNA Fragment Purification
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DNA fragments were PCR amplified from plasmids in 800 μl volumes using a Q5 High-Fidelity PCR kit (NEB) following the manufacturer’s recommended procedure. After amplification, the reaction was then passed through a single column from a Monarch PCR & DNA cleanup kit (NEB) following the manufacturer’s recommendations for double-stranded DNA <2 kb. The column was eluted with 15 μl elution buffer and digested with 10 U AvaI at 37 °C for 2 h. For KLHL15 promoter fragments only, before digesting with AvaI, the eluate was digested with 10 U SmaI at 37 °C for 1 h followed by treatment with 5 U QuickCIP (NEB) for 30 min at 37 °C and heat inactivation for 10 min at 80 °C. The reactions were stopped by adding 4 μl 6× Purple Gel Loading Dye (NEB) and then run on a 0.8% Tris:acetate:EDTA agarose gel. Fragments were gel purified using a QIAquick gel extraction kit (Qiagen) according to the manufacturer’s recommendations and were further concentrated by ethanol precipitation.
4
Plasmid DNA Ligation Kinetics
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Reaction mixtures (10 μl) contained 40 ng of Xbal linearized (cohesive ends) pUC19 plasmid, 10 mM MgCl2, 1 mM ATP, 150 mM NaCl, 50 mM TrisHCl 7.5, 1 mM EDTA, 1 mM DTT, 2 μM X4L4 or 8 U/μL T4 DNA ligase (New England BioLabs) and an increasing amount of PEG 8k (w/v) as indicated. 2 μM XLF were added when indicated. The reactions were incubated at room temperature for 5 minutes. Then, the mixtures were deproteinized by adding pronase E (4 μg/μl) and 1X purple gel loading dye (New England BioLabs) and incubated at 55°C for 30 minutes. The reaction products were analyzed by 0.8% agarose gel electrophoresis using TBE buffer and stained with SYBR Green I. Gel images were visualized and quantified by Gel Doc XR+ and Image Lab™. Each reaction was carried out in triplicate. The reactions were also monitored by a fluorescence microscope with a 60X magnification objective using similar conditions but without adding ATP and MgCl2.
5
M13mp18 ssDNA Cleavage Assay
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The DNA cleavage assays were performed as previously described (17) . Briefly, 2 nM M13mp18 circular ssDNA (New England Biolabs) (Table S3) was treated with 200 nM LlCsm complex, 200 nM target RNA at 37 C/10 to 90 min as indicated in a cleavage buffer containing 33 mM Tris acetate pH 7.6/32 C, 66 mM potassium acetate, 10 mM MnCl 2 . The reactions were quenched using 1x purple gel loading dye (New England Biolabs). The reaction products were heated at 95 C/5 min and separated on 1% agarose gel in Tris/Acetic acid/EDTA (TAE) running buffer and were visualized by staining with Ethidium Bromide.
6
Gel Electrophoresis of PCR and Digestion
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To analyse the PCR and digestion products, 1% (w/v) of agarose (Sigma-Aldrich, USA) was prepared using 0.5 × TBE buffer (45 mM Tris–borate and 1 mM EDTA), Invitrogen SYBR® Safe DNA Gel Stain (1:10,000 dilution), and run at 100 V for one hour. Purple Gel Loading Dye (New England Biolabs, UK) was used to load the samples into the gel and 1 kb Plus DNA Ladder (New England Biolabs, UK) for analysis.
7
Visualizing Biotinylated DNA-Streptavidin Complexes
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Randomly biotinylated DNA templates were prepared as described above except that an unmodified reverse primer (Supplementary Table S1 , oligonucleotide K) was used. 1 pmol of DNA template was incubated in the absence or presence of 10 pmol SAv for 15 min at room temperature. Six times Purple Gel Loading Dye (no SDS) (New England Biolabs) was added and samples were fractionated in using a 1.5% Agarose gel containing 1× GelRed (Biotium). Bands were detected by UV transillumination using a ChemiDoc (Bio-Rad).
8
CX3CR1 Nucleosome Binding Assay
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180bp native CX3CR1 nucleosome (Table S2) was used in this assay. 1000 ng nucleosome was mixed with PU.1 at different concentrations in 18.5 μl reaction system in buffer 20 mM HEPES, pH 7.1, 100 mM NaCl and 0.5 mM TCEP. After 20 min incubation at room temperature, 1 μl Cutsmart buffer and 1 U Sau96I (New England Biolabs) were added into the system. The mixture was placed into 37 °C incubator for 20 min. Purple gel loading dye (New England Biolabs) was added to stop the enzyme digestion reaction. Then 1 μl proteinase K (New England Biolabs) was used at 50 °C for 1 hour to digest PU.1 and histones. 4 μl of the digestion products were analyzed on 5 % acrylamide gels in 0.2 × TBE at 120 V for 60 minutes at 4 °C. After electrophoresis, gels were stained with ethidium bromide (EtBr) and quantified using ImageJ. Three independent experiments were performed. Microsoft Excel and Prism were used to generate the figures.
9
Plasmid Size Determination by Gel Electrophoresis
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To determine the size of plasmids, a 0.8% agarose (MIDSCI) gel was run at 120 V for ~40 min on an electrophoresis equipment (Bio‐Rad). Before electrophoresis, extracted plasmids were mixed with the Purple Gel Loading Dye (6×, no SDS; New England Biolabs) and loaded into wells in the agarose gel. Quick‐Load® Purple 1 kb DNA Ladder (New England Biolabs) or Quick‐Load® 1 kb Extend DNA Ladder (New England Biolabs) was used for the reference of DNA length. Gel images were inverted by ImageJ for visualization purpose.
10
Topoisomerase I-Mediated Catenane Topology
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Catenated DNA molecules (catenated products, CP) were from Twister Biotech, Inc. (Houston, TX, USA). These DNA catenanes consist of a 3530 bp large circular DNA (large circle, LC) linked to a 339 bp minicircle (mC) and are generated by λ–integrase-mediated site-specific recombination on the parent plasmid (PP) pMC339 (35 (link)). As controls, three different DNA topological forms—supercoiled, linearized or nicked- of these multi-linked catenanes were obtained by incubating the DNA with the restriction enzymes BamHI or NdeI, or the nicking endonucleases Nt.BspQI or Nb.BbvCI, respectively. The superhelical density of the supercoiled multi-linked DNA was −0.07 (35 (link),36 (link)). TopA was incubated in 10 μl TR buffer containing 10 mM MgCl2 and 0.27 μg of catenated DNA at various (as indicated) temperatures for 20 min. Reactions were stopped by cooling the reaction mixture on ice for at least 10 min. A total of 0.1% SDS (final concentration) with Purple Gel Loading Dye (no SDS) (from NEB) was added before loading onto gels. The reaction products were analyzed on 1.7 or 3.5% agarose gels in TEP buffer at room temperature and run at 3 V/cm for 2–4 h. The gels were stained by incubation in TEP buffer containing 2 μg/ml ethidium bromide for 30 min and stored in water before digitalization.
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