I'm facing challenges with the purity of plasmid DNA extracted from Trypanosoma brucei 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. I think you need to confirm the contamination issue to exclude other causes. what about the 260/280 ratio? do you perform GAPDH or Beta actin amplification on the same samples to ensure extract DNA integrity? If yes, try to spike ur positive control into one of the pre-extract samples. After this, apply extraction, and see if there are problems in the resulting spiked samples. General speaking, increased washing steps is recommended if you really face impurities issue. one more thing, Internal positive control for other targets is a good idea for your problem as well.
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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