Back to basics: Important things to keep in mind when purifying plasmids and DNA fragments

  • DNA binds to silica under high salt conditions, and releases from silica under low salt conditions.
    This is why binding buffers are made with salts and DNA elution buffers do not contain salt.

  • Larger fragments are more difficult to elute because they bind more tightly to the column’s matrix.
    Therefore, they are more challenging to release from the column. Warming the elution buffer and also allowing the elution buffer to sit on the column can increase the efficiency of elution.

  • When carrying out plasmid minipreps, prepping more cells does not necessarily equate to more plasmid DNA recovery.
    Using too much cell culture can clog the column and reduce binding efficiency, actually lowering your recovery efficiency. There is usually an ideal culture OD range that is optimal for a given column. See our Do’s and Don’ts of Minipreps and our Guidelines for Purifying Low Copy Plasmids for more useful tips.

  • When working with low copy plasmids, it is not uncommon to see residual gDNA in your plasmid preps.
    Exonuclease V (RecBCD) can be used to remove gDNA, as it selectively digests linear DNA, and leaves circular DNA intact. Check out this application note for more information.

  • Residual ethanol in your eluate is common if using columns that contain retaining rings (plastic rings to hold the membrane in place).
    Ethanol in your eluate can interfere with downstream applications and also can cause your sample to float out of the well when loading your gel lanes. Monarch DNA Cleanup Columns and Monarch Plasmid Miniprep Columns are designed without retaining rings, thereby eliminating the risk of carryover contamination.

  • A very common reason that people experience low yields when carrying out gel extraction is that they do not properly release all of the DNA from the agarose gel.
    It is important to use the recommended amount of dissolving buffer and to ensure that the agarose is completely melted before proceeding. For more tips on gel extraction, see our Six Tips for Perfect Gel Extraction.

Learn more about Nucleic Acid Purification products from NEB at