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Further Purification Steps needed
Hello. I have been using Qiagen DNAeasy Blood and Tissue Kits to extract prey DNA from the guts of their predators. There seems to be inhibatory substances present in my DNA samples even after using this extraction method. I ran tests by placing DNA solutions testing positive for prey remains with those that tested negative and the negative solutions always caused the positive solutions to become negative.
I optimized my PCR reactions using 25ng/ul of my prey DNA, which were diluted from stocks ranging from 50-120ng/ul. After DNA extraction from my predator's guts DNA concentrations range from 450-600ng/ul. I cannot dilute these solutions or risk not detecting prey DNA when predation occurred. I tried adding different %v/v of BSA to no avail. I know qiagen also makes DNA stool extraction kits that contain InhibitEX tablets which are supposed to bind to inhibators found in fecal matter, but I don't know what inhibators are present.
Are there any additional DNA purification steps I could try after using Qiagen kits? Im not sure exactly what inhibitors are present. My predators feed on the phoem tissues of trees so there could be complex polysaccharides, and since I'm using the digestive tracts of my predators there could be inhibators found there as well.
Any advice is greatly appreciated.
|needed , purification , steps|
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