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The Ct values are close to the negative because we were extracting the DNA in an inappropriately way. It is our fault. It is not a matter of the MasterMix. The mix works perfectly. We are using the SensiFAST Probe Lo-ROX Kit from Bioline.
Following your advice, today we repeated the DNA extraction leaving a little bit of the aqueous phase with the organic phase and the intermediate layer. To our surprise and happiness, the results were much better than the previous ones. We were able to detect DNA from bacteria infecting seeds at less than 1 UFC/ml of bacteria (we use 10 ml as starting material, which means approx. 7 cell per extraction tube).
And, What was the difference? this time we kept the interphase with the organic phase. In previous experiments during the DNA isolation we removed the interphase or part of it. If we keep this intermediate phase we get a final pellet very big (DNA and other remains) and not very clean. The samples that we process are from seeds (difficult samples) and we thought that the intermediate layer could contain PCR inhibitors. So, we decided to remove it. This time we decided to retain the intermediate layer with the organic phase to isolate the DNA. Things were much better and no PCR inhibitors were detected. Probably, we were losing a lot of DNA by removing the interphase.
The today results looks very promising even with difficult samples as seeds.
I want to thank you the time you have spent trying to solve our problem. Your help has been very valuable for us.
Dr Leandro de Leon, Anove Asociación
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