Working with low copy plasmids is often challenging due to the reduced yield after purification from bacterial cultures. These plasmids utilize origins of replication (ORI) sequences that are maintained at much fewer copies of the plasmid per cell (15-20) than higher copy plasmids like pUC19 and derivatives. While useful for maintaining the integrity of long DNA or reducing the toxicity of cloned sequences, recovery of low copy vectors is a pain point in molecular biology workflows. Many researchers assume that simply using more cells will help improve yield, but in fact, this is not always true. In some cases, using more cells can overload the column or overwhelm the buffer system and can actually result in a much lower yield and reduced purity. Additionally, users of low copy plasmids often see contaminating host strain genomic DNA in their preps because there is such a high proportion of gDNA to plasmid DNA. While these challenges cannot always be solved, below are some guidelines and tips to increase purification yield when working with low copy plasmids.
Do not overgrow the culture. Cells that are overgrown can lyse in culture, reducing plasmid yield because plasmid no longer remains in the bacterial pellet used for the prep. It is ideal to harvest the culture near the end of log phase growth (often, 12-16 hours after inoculation).
- Do not overload the column. Using too many cells in a single prep may overload the column and result in low yield. Excess biomass reduces the lysis efficiency of the buffer system and does not allow all the genomic DNA to complex with the debris formed during the neutralization step. Additionally, the excess biomass and debris may not pellet well and can clog the column if transferred during the binding step. Lastly, the larger cell lysate that results from using more cells has higher RNA amounts and care must be taken to ensure the RNase A in the neutralization buffer has time to clear this RNA burden. We recommend using no more than 5 ml or 15 OD units of culture when working with E.coli strains carrying low copy plasmids. Users can also split a larger bacterial pellet and run two parallel preps that are pooled after elution for improved performance, if using more cells is required.
- If you see genomic DNA, consider treatment of the purified plasmid with Exonuclease V (RecBCD). This enzyme (NEB #M0345) selectively digests linear DNA (genomic DNA/gDNA) while leaving circular DNA intact (plasmid DNA). We outline the specific protocol in our application note.
- Consider Chloramphenicol Amplification. This “old school” technique is outlined by Maniatis, Fritsch, and Sandbrook in their Molecular Cloning: A Laboratory Manual. This method allows plasmids containing the pMB1 or ColE1 origins to be amplified by stopping cellular growth by adding chloramphenicol to 170 mg/L to arrest ribosomal function. This allows host encoded enzymes, including DNA Pol I to copy the plasmids already in the cytoplasm, thereby increasing the yield of recovered plasmid after purification. If you perform the chloramphenicol amplification culture modification, be sure to not use more than 15 OD units of cells for your miniprep. For details please refer to standard manuals for cloning procedures (e.g., Molecular Cloning : A laboratory manual / T. Maniatis, E.F. Fritsch, J. Sambrook).