A gel-less isolation of untagged plasmid DNA insert from vector backbone in homogeneous format

Agarose gel electrophoresis with subsequent DNA extraction from gel is routinely used for DNA fragment isolation after plasmid DNA digestion. We describe a gel-less method for DNA fragment isolation after plasmid DNA digestion which is based on in-solution negative selection through depletion of vec...

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Bibliographic Details
Published in:Analytical biochemistry 2017-03, Vol.521, p.28-30
Main Authors: Kalinichenko, Svetlana V., Shepelev, Mikhail V., Korobko, Igor V.
Format: Article
Language:English
Subjects:
Animals
Animals, Genetically Modified
DNA - genetics
DNA - isolation & purification
DNA fragment isolation
Genetic Vectors
Homogeneous format
Integrases - genetics
Negative selection
Plasmid DNA digestion
Plasmids - genetics
Plasmids - isolation & purification
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Summary:Agarose gel electrophoresis with subsequent DNA extraction from gel is routinely used for DNA fragment isolation after plasmid DNA digestion. We describe a gel-less method for DNA fragment isolation after plasmid DNA digestion which is based on in-solution negative selection through depletion of vector backbone bearing LoxP sites by sorption on solid phase-immobilized mutated Cre recombinase. The method might be especially useful in preparation of DNA fragments for transgenic animal generation where residual agarose presence is a concern, and DNA fragments are frequently large in size and thus might be mechanically damaged during purification with conventional affinity-based gel extraction methods.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2017.01.006