Nuclease Contamination
Nuclease Contamination - Related Videos
07:50
Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase
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Cited by 10 •
2017
Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.
12:04
Mouse Genome Engineering Using Designer Nucleases
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Cited by 14 •
2014
Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.
01:16
Contaminants and Errors
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2024
Effective sample preparation is crucial for accurate and reliable laboratory analysis. During this process, two significant sources of error can arise: concentration bias from improper sample splitting and contamination caused by methods used to reduce particle size, such as grinding or homogenization. Identifying and minimizing these potential errors is crucial to ensuring the validity of the analysis. Another key consideration is determining the appropriate number of samples required to...
08:13
Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
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2025
This article describes a methodology for overexpressing recombinant Nsp15, a toxic nuclease, in a C41(DE3) expression system, followed by purification of the tagged protein utilizing affinity and size exclusion chromatography. These protocols can be adapted for other challenging toxic proteins.
07:42
Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)
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Cited by 16 •
2016
Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.
FAQs
The critical chemical event is cleavage of phosphodiester bonds in the nucleic-acid backbone. DNases act on DNA, whereas RNases act on RNA, so the affected molecule depends on the contaminating enzyme. Continued cleavage can convert intact genetic material into fragments or eliminate it entirely, reducing the amount of usable template available for downstream molecular biology experiments.
The nucleic acid being studied determines which contamination risk is most consequential. DNA-focused work can be impaired by DNases, while RNA-focused work can be impaired by RNases. This distinction helps researchers select suitable nuclease-free reagents, maintain appropriate handling practices, and interpret degradation-related problems in applications such as PCR, sequencing, cloning, or gene expression analysis.
Possible consequences include fragmented genetic material, complete loss of the target nucleic acid, weak analytical signals, or a failed reaction. These effects can interfere with amplification, sequencing, cloning, and gene expression measurements. Because contamination may alter the available template rather than the intended biological system, it can also produce misleading results instead of simply an obvious experimental failure.
Use clean materials and nuclease-free reagents throughout handling and analysis. These measures reduce opportunities for degradative enzymes to contact the sample before the experiment is complete. Appropriate controls should accompany the workflow so that unexpected weak signals or failed reactions can be evaluated against evidence from the handling and reagent conditions used.
Controls provide a comparison for judging whether weak signals, failed reactions, or other unexpected outcomes reflect compromised nucleic acid integrity. When controls are included alongside samples, researchers can assess the reliability of the workflow rather than interpreting one affected result in isolation. This is especially important for PCR, sequencing, cloning, and gene expression studies.
Different applications depend on preserving genetic material for distinct analytical purposes, but all can be affected when degradation changes the available DNA or RNA. PCR may show weak signals or fail, while sequencing, cloning, and gene expression studies may yield compromised or misleading results. Preventing contamination therefore supports dependable interpretation across molecular biology and biotechnology workflows.