Removal of contaminating bacterial chromosomal DNA in plasmid preparations (but also cosmid, BAC…) is crucial in today’s precise molecular biology experiments. In this post, a cost-effective but powerful enzyme is described for molecular biologists looking for genomic DNA-free preps.
DNA from plasmid, cosmid, fosmid, and BAC clones or vector preparations are frequently contaminated with fragments of bacterial genomic DNA generated during alkaline lysis. These contaminations lead to false positives clones, high backgrounds, or erroneous sequence data.
Various purification methods are available but they do not effectively remove all genomic DNA traces. Additional purification steps are then required. For this purpose, you might consider the use of the Plasmid-Safe™ ATP-Dependent DNase (Epicentre, an illumina company) as a fast and easy method to efficiently elimnate genomic DNA with minimal handling time.
Plasmid-Safe™ ATP-Dependent DNase selectively hydrolyzes linear double-stranded DNA to deoxynucleotides at slightly alkaline pH and, with a lower efficiency, linear and closed-circular single-stranded DNAs. Interestingly, this DNase does not affect closed-circular supercoiled or nicked circular double-stranded DNA, and is conveniently and completely heat-inactivated by a 30 minute incubation at 70°C.
These features make the Plasmid-Safe™ ATP-Dependent DNase an ideal cost-effective approach for the final purification stages for plasmid, cosmid, fosmid, and BAC vector and clone preparations. This final “cleanup” avoids the problems caused by contaminating genomic DNA and ensures highly pure closed-circular supercoiled or nicked circular double-stranded DNA preps for locus-specific TALE genome editing (1, 2), RiboFR-Seq (3), and studies for oversized Adeno-Associated Viral vector preps (4).
- Ma A.C. et al. “FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science” (2016) Human Gene Therapy, Vol. 27. DOI: 10.1089/hum.2015.172
- Ely A. et al. “Progress With Developing Use of Gene Editing To Cure Chronic Infection With Hepatitis B Virus” (2016) Molecular Therapy. DOI : 10.1038/mt.2016.43
- Kyostio-Moore S. at al. “The impact of minimally oversized adeno-associated viral vectors encoding human factor VIII on vector potency in vivo” (2016) Mol Ther Methods Clin Dev, Vol. 3. DOI : 10.1038/mtm.2016.6
- Zhang Y. “RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes” (2016) Nucl Acids Res .DOI : 10.1093/nar/gkw165
- CircLigase™ ssDNA Ligase is an ATP-dependent ligase catalyzing intramolecular ligation, also known known as circularization, of ssDNA templates with a 5′-phosphate and a 3′-hydroxyl group in the absence of a complementary sequence. The enzyme is therefore useful for making circular ssDNA molecules from linear ssDNA for rolling-circle replication, rolling-circle transcription, ligation step in RNA-Seq library preparation (eg. NET-Seq).
- QuickExtract™ DNA Extraction Solution (Epibio – Illumina) enables rapid and efficient PCR-ready genomic DNA extraction from almost any sample type (eg. hair follicles, tissue-culture cells, buccal cells, zebrafish organs, mouse tail snips…) in less than 8 minutes. You can rapidly and efficiently extract PCR-ready DNAs for simultaneous analysis of one up to hundreds of DNA samples for genotyping and genetic studies.
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Why not outsource your DNA preps?
It’s now possible to outsource DNA Plasmid preps. These preparations, validated and ready-to-use, can be obtained within only a few days through tebu-bio. To know more, read the post dedicated to this topic written by Frederic.