1. Nicking or Linearization: Cause: Plasmid DNA may undergo nicking or complete linearization due to mechanical shear forces during extraction or handling, or enzymatic action by nucleases. Solution: Ensure gentle handling of the DNA during purification and minimize pipetting or vortexing. Use nuclease-free reagents and containers to prevent enzymatic degradation.
2. Topoisomerase Activity: Cause: Topoisomerases present in bacterial cultures or contamination in reagents can relax supercoiled DNA. Solution: Employ DNA purification kits that effectively remove proteins, including topoisomerases, from the plasmid preparation.
3. Extraction or Purification Method: Cause: The choice of plasmid DNA extraction or purification method may not be optimal, potentially leading to relaxed or degraded DNA. Solution: Opt for a high-quality plasmid DNA purification kit, preferably one with a column-based purification step that yields high-quality, supercoiled DNA. Ensure that the protocol is followed meticulously.
4. Storage Conditions:Cause: Improper storage conditions (e.g., repeated freeze-thaw cycles, storage at inappropriate temperatures) can lead to the degradation or alteration of plasmid DNA. Solution: Store plasmid DNA at -20°C or -80°C in a suitable buffer, and avoid multiple freeze-thaw cycles by aliquoting.
5. Agarose Gel Electrophoresis Analysis: Cause: Incorrect interpretation of agarose gel results can lead to the mistaken assumption that DNA is not supercoiled. Supercoiled DNA migrates faster than linear or open-circular forms but can appear in multiple forms depending on the gel concentration and voltage applied. Solution: Use appropriate gel concentration and electrophoresis conditions to resolve supercoiled DNA distinctly from other forms. Consider using a reference supercoiled plasmid as a control.
6. pH and Ionic Conditions: Cause: Suboptimal pH and ionic conditions during purification, storage, or analysis can affect the supercoiled state of plasmid DNA. Solution: Ensure that buffers used during purification, storage, and analysis are at the correct pH and ionic strength to maintain DNA integrity.

Conclusion

In conclusion, the absence of apparent supercoiling in plasmid DNA preparations can result from a variety of factors related to DNA extraction, purification, handling, and analysis. By systematically addressing each potential cause and implementing the corresponding solutions, you can enhance the likelihood of obtaining and maintaining plasmid DNA in its supercoiled form.