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Updated: May 14, 2026
Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks
Published on: November 25, 2015
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.
Insights
Plasmid DNA purification using miniprep kits can lead to RNase contamination, impacting experiments. Traditional cesium chloride (CsCl) density gradient methods offer a superior, RNase-free alternative for highly pure plasmid DNA.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Miniprep and maxiprep kits are widely used for plasmid DNA preparation.
- Plasmid DNA prepared with these kits is often used for in vitro transcription and cell transfection.
- Contamination with RNases, particularly pancreatic RNase, is a significant issue with commercial DNA preparation kits.
Purpose of the Study:
- To highlight the problem of RNase contamination in plasmid DNA prepared using commercial kits.
- To recommend an alternative purification method for obtaining highly pure plasmid DNA.
- To provide a reliable protocol for RNase-free plasmid DNA suitable for sensitive downstream applications.
Main Methods:
- Evaluation of plasmid DNA purity from miniprep/maxiprep kits.
- Comparison with plasmid DNA purified using traditional cesium chloride (CsCl) density gradient ultracentrifugation.
- Assessment of RNase contamination in DNA preparations.
Main Results:
- Miniprep and maxiprep DNA frequently contains difficult-to-inactivate RNase contaminants.
- Cesium chloride (CsCl) density gradient methods yield highly pure, RNase-free plasmid DNA.
- The described CsCl protocol provides sufficient DNA for multiple experiments.
Conclusions:
- Traditional CsCl density gradient purification is recommended over commercial kits for plasmid DNA intended for transcription or transfection.
- Investigative efforts should focus on RNase-free DNA purification to ensure experimental reproducibility.
- The CsCl method provides a robust and scalable approach for obtaining high-quality plasmid DNA.
