The PCR work very well with Parasite DNA, but, when the Parasite DNA is added to the soil samples and the DNA is extracted from contaminated soil samples, to used in the same PCR, the PCR is inhibited.
When you extract DNA from soil you will co-extract humic substances that will bind to the reagents and DNA in your reaction and inhibit the PCR. If you haven't used a specialized soil extraction kit, this is the first thing I would try. I have used one called FastDNA spin kit for soil from MP biomedical. This removes some of the humic compounds, but for me it didn't completely do the trick so I used an additional kit to purify the samples after extraction. The one I used is called Onestep pcr inhibitor removal kit from Zymo research.
You can also try and dilute your samples something like 10-100x before using them for PCR to reduce the inhibition.
Dear Elizabeth,
1. I didn't understand the part of adding DNA to soil sample. Are you adding the intact parasite or just the isolated DNA to the soil?
2. If you are adding the DNA directly to the soil sample, then likely the environmental nucleases (in your case DNases) act upon your DNA, later when you isolate it and try amplify it, may be there isn't proper template left in the soil.
3. After adding DNA to soil, when you isolate it again, do you check its quantity and quality in gel electrohoresis? If DNA is intact in the isolated sample, but your PCR is failing, then may be along with the isolated DNA there are contaminants from the soil which are inhibiting the PCR reaction. How do you isolate your DNA?
It would be better if you can attach a picture of your gel with the intact parasite DNA PCR product and the PCR product afterwards.
When you extract DNA from soil you will co-extract humic substances that will bind to the reagents and DNA in your reaction and inhibit the PCR. If you haven't used a specialized soil extraction kit, this is the first thing I would try. I have used one called FastDNA spin kit for soil from MP biomedical. This removes some of the humic compounds, but for me it didn't completely do the trick so I used an additional kit to purify the samples after extraction. The one I used is called Onestep pcr inhibitor removal kit from Zymo research.
You can also try and dilute your samples something like 10-100x before using them for PCR to reduce the inhibition.
Hi, Elizabeth,
As Karin said, soil samples are plenty of PCR inhibitors, like humic and tanic acids, complex polysacharides from plants and many others. If a soil specific DNA extraction kits does not work with your samples you can also try with a stool DNA extraction kit. Stools are also samples that carry a lot of PCR inhibitors and there are several extraction kits designed to remove them.
Hi Ellizabeh
a PCR can be inhibited when working with soil samples because there is an interaction between the ions magnesium and humic acids
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