NucleoSpin® Plasmid EasyPure

Protocol tips

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	Starting material from 1–5 mL cultures

Protocol tips
Starting material from 1–5 mL cultures

Publication protocol

Plasmids were isolated from 2 mL of C. difficile overnight culture using NucleoSpin Plasmid Easypure columns (Macherey-Nagel); to increase yield, 10 mg/mL lysozyme to buffer A1 was added, as recommended by the manufacturer. Using PCR and sequencing, we found that the DNA isolated using this kit is heavily contaminated with chromosomal DNA. To isolate pure plasmid DNA, aliquots of the DNA were incubated with PlasmidSafe ATP-dependent DNase (Epicentre) that digests linear, but not circular double stranded DNA. After purification with a Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel), the absence of genomic DNA was confirmed by PCR using primers targeting gluD. Yields were generally very low, but the plasmid was readily detectable by PCR.

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Manufacturer protocol

Download the product protocol from Macherey Nagel for NucleoSpin® Plasmid EasyPure below.

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