Plasmid Purification Troubleshooting
Plasmid DNA purification, better known as plasmid preparation, is one of the most commonly used methods in the lab. The preparation includes; the growth of a bacterial culture, the harvesting, and lysis of bacterial cells and the purification of plasmid DNA. The purified plasmid DNA is further used in downstream processes such as; cloning, gene transfer, transformation or sequencing. Several steps can be improved to get higher yields or to get rid of contaminations within the final product.
If you are experiencing an issue, don't worry! The ZAGENO troubleshooting guide is here to help.
Incomplete cell lysis
Cell density is too high: Reduce culture volume.
Cell pellet not completely resuspended: Use an appropriate buffer or solution to properly resuspend the cell pellet prior cell lysis.
Insufficient alkaline lysis step: Check the lysis solution for salt precipitates.
Contaminations with DNA or RNA
Genomic DNA contamination: Do not vortex the cells too vigorously during the lysis and neutralization step.
Lyophilized RNase not completely dissolved: Dissolve the RNase as mentioned in the protocols.
RNA contamination: Add RNase to the resuspension solution before first use.
Too many bacterial cells: Reduce culture volume so as to not overload the reaction or column.
Cultures are too old: Do not use cultures cultivated for more than 24 hours or that are already in the death phase.
Low or no yield
Culture volume too high: Reduce culture volume to increase the number of lysed cells.
Plasmid did not propagate: Use freshly streaked bacterial cells for inoculation.
Selective pressure too low: Use an appropriate quantity of antibiotics during the cultivation to avoid overgrowth of non-transformed cells without the plasmid of interest.
The culture was not processed immediately: Always use a freshly grown culture. If you need to process the preparation another day; pellet the cells, remove the culture medium and then store the cell pellet at -70 °C.
Reduced effectiveness of kit components: Store the kit components under appropriate conditions.
Wash solution too concentrated: Avoid evaporation of ethanol by keeping the bottle tightly capped.
Poor performance in downstream processes
Additional plasmid forms included: Avoid denaturation of the plasmid DNA - do not prolong cell lysis for more than recommended in the protocol.
Plasmid DNA concentration is too low: Precipitate the plasmid DNA and resuspend in a smaller volume to concentrate the DNA.
Ethanol present in the resuspended plasmid DNA: Increase the drying time.
Plasmid Puification Tips
During plasmid preparation, make sure to use a salt such as sodium acetate to help precipitate the nucleic acids out of solution.
- The positively charged sodium ions react with the sugar-phosphate backbone of the plasmids, making the plasmid more hydrophobic and less soluble in water.
- Sodium acetate is particularly effective in DNA plasmid purification
- Use lithium chloride as the salt for purification of RNA plasmids
During plasmid preparation, make sure to use ethanol as the solvent if the sample is small, its volume is sufficient, or if longer storage time is required.
- Both nucleic acids and salts are less soluble in isopropanol than ethanol.
- If you can only fit 1 volume of alcohol to 1 volume of your DNA sample, use isopropanol instead
During plasmid preparation, make sure to incubate the DNA, sodium acetate, and ethanol mixture at a temperature of at least -4 °C for approximately 20 minutes. However, -20 °C or -40 °C is preferable.
- If the yield is low, try increasing the incubation time to 30-60 minutes
Do not use too much biomass for the plasmid preparation.
Always use freshly grown cultures.
Avoid air bubbles during the resuspension of the cell pellet.
Do not prolong the incubation time within the lysis step.- Dashes work just as well