I'm facing challenges with the purity of plasmid DNA extracted from shigella for PCR applications. Despite using a commercial DNA extraction kit, I suspect the presence of contaminants or impurities that might be affecting my PCR results. What are the best practices and specific steps I should follow to guarantee the highest level of purity in my plasmid DNA samples?
Hello,
You can elute the plasmid DNA with sterile distilled water. The commercial elution buffer contains some chemicals which may inhibit PCR.
Do not load too much of plasmid DNA as it is more efficient than the genomic DNA.
Also, standardized the PCR protocol before using new samples (for instance, using a gradient temperature PCR to optimize the annealing temperature).
Best regards.
You don't need extremely pure DNA for PCR, that is why you can do PCR on environmental and clinical samples and it works. If you think you have inhibitors in your sample, try using less sample. Especially with plasmids we often use vast excess of template relative to what is required.
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