Three hundred (300) bp DNA targets containing the target sequence ~200 bp from one end and the flanking genomic context were synthesized by Twist Bioscience, PCR amplified using the provided universal primers, purified, and resuspended in nuclease-free water to 100 nM. Three technical replications of reactions were assembled in the following order: 7 μL nuclease-free water, 1 μL target DNA substrate (100 nM), 1 μL 10x Cas9 Nuclease Reaction buffer (200 mM HEPES, 1 M NaCl, 50 mM MgCl2, 1 mM EDTA (pH 6.5 at 25°C)), 1 μL Cas9-RNP (1 mM), then incubated for 1 hour at 37°C followed by proteinase K digestion (1 μL - 56°C for 10 minutes; ThermoFisher, #EO0491). Products were resolved on a 3% agarose gel stained with SYBR Gold and analyzed using ImageJ.
Cas9 nuclease reaction buffer
Manufactured by Thermo Fisher Scientific
The 10x Cas9 Nuclease Reaction buffer is a solution designed to facilitate the enzymatic activity of the Cas9 nuclease, a key component in CRISPR gene editing systems. This buffer provides the optimal ionic conditions and cofactors required for Cas9 to effectively bind and cleave target DNA sequences. The 10x concentration allows for easy dilution and use in various experimental setups.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using cas9 nuclease reaction buffer
1
Cas9-Mediated DNA Cleavage Assay
2
Cas9-mediated DNA Cleavage Assay
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Three hundred (300) bp DNA targets containing the target sequence ~200 bp from one end and the flanking genomic context were synthesized by Twist Bioscience, PCR amplified using the provided universal primers, purified, and resuspended in nuclease-free water to 100 nM. Three technical replications of reactions were assembled in the following order: 7 μL nuclease-free water, 1 μL target DNA substrate (100 nM), 1 μL 10x Cas9 Nuclease Reaction buffer (200 mM HEPES, 1 M NaCl, 50 mM MgCl2, 1 mM EDTA (pH 6.5 at 25 °C)), 1 μL Cas9-RNP (1 mM), then incubated for 1 hour at 37 °C followed by proteinase K digestion (1 μL - 56 °C for 10 min; ThermoFisher, #EO0491). Products were resolved on a 3% agarose gel stained with SYBR Gold and analyzed using ImageJ.
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