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Cold Spring Harb Protoc. 2013 Feb 1;2013(2):174-9. doi: 10.1101/pdb.prot072926.

RNA-friendly plasmid preparation.

Abstract

With the advent of miniprep kits for the preparation of plasmids, we have noticed that many investigators use plasmids prepared by these methods as transcription templates or for cell transfection experiments. This can be quite problematic because miniprep (or maxiprep) DNA can often be contaminated with RNases. Indeed, most DNA preparation kits include pancreatic RNase. This RNase is extremely difficult to inactivate and can contaminate benchtops and pipettors. We highly recommend that plasmid DNAs, especially those that will be used repeatedly, be purified using traditional cesium chloride (CsCl) density gradient methods that do not involve the use of RNase. We have found that the protocol described here yields highly pure, noncontaminated plasmid DNA suitable for in vitro transcription and/or transfection analyses. An added benefit to this procedure is that it will yield enough plasmid DNA for many, many experiments.

PMID:
23378655
DOI:
10.1101/pdb.prot072926
[Indexed for MEDLINE]
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