Plasmid DNA Preparation Troubleshooting

Extracted from Nucleic Acid Sample Preparation for Downstream Analyses, GE Healthcare, 2007

Table 5.6 Possible causes and solutions for problems that may occur during plasmid DNA preparation. These troubleshooting tips are applicable to the majority of commercially available plasmid DNA purification kits.

Problem	Possible cause	Solution
Yield of plasmid DNA is low	The bacterial culture was not fresh.	A culture should be processed in a timely manner after it has reached the required cell density. Alternatively, bacterial pellets can be stored at -20 °C prior to plasmid DNA extraction with no significant effect on purity or quality.
	The plasmid has a low copy number.	Process a larger volume of culture or grow in a richer medium. Alternatively, clone DNA sequence of interest into a plasmid possessing a higher copy number, for example, pCORON1000 (~300–500 copies per cell).
	The purification system was overloaded.	Measure the A600 of the culture before processing to ensure that the purification column is not overloaded with bacterial debris (e.g., when using illustra™ plasmidPrep Mini Spin system. If the A600 is > 5, reduce the volume of culture processed).
	Cells were not adequately resuspended.	Cell resuspension can be achieved by either vortexing, pipetting up/down, or running the microcentrifuge tube containing the bacterial cells along the lid of an empty pipette tip box.
	Cells were not adequately lysed.	Mix gently but completely when alkaline lysis solution is added.
	EndA+ strains (e.g., HB101) possess nucleases and excessive carbohydrates.	When using silica-based purification formats, apply an additional wash to the plasmid purification column (after plasmid DNA binding) using high-salt buffer. This additional step removes residual nucleases and excessive carbohydrates, thereby increasing the yield and quality of the plasmid DNA. Alternatively, use an EndA- strain, such as TOP10 or DH5α.
Plasmid DNA appears as a smear when viewed on an agarose gel	EndA+ strains (e.g., HB101) possess nucleases.	When using silica-based purification formats, apply an additional wash to the plasmid purification column (after plasmid DNA binding) using high-salt buffer. This additional step removes the residual nuclease activity, thereby increasing yield and quality of the plasmid DNA. Alternatively, use an EndA-  strain such as TOP10 or DH5α.
Plasmid DNA is contaminated with genomic DNA	Alkaline lysis: The sample was mixed too vigorously after adding lysis buffers.	Mix gently by inverting the sample 10–15 times after adding either of the solutions. Vigorous mixing may cause shearing of genomic DNA, thereby facilitating its copurification with plasmid DNA.
Plasmid DNA is contaminated with RNA	Sample was not treated with RNase.	RNase (100 μg/mL) is included in the lysis buffer of illustra™ plasmidPrep Mini Spin Kit. When using other methods, add an RNase step if it is not already included.
Plasmid DNA does not cut to completion at 37 °C	Plasmid DNA was irreversibly denatured and therefore will not cut.	Mix gently by inverting the bacterial sample 10–15 times after adding the alkaline lysis solution. Avoid prolonged exposure (> 5 min) or the NaOH in the lysis solution will irreversibly denature plasmid DNA.
	Silica-based purification: All traces of ethanolic wash buffer were not removed.	The presence of residual ethanol may affect downstream applications and therefore it must be carefully removed, for example, after washing and drying the purification column by centrifugation. If any ethanolic wash buffer comes into contact with the purification column after centrifugation, discard the flowthrough and recentrifuge.