I have been trying to amplify a gene from DNA extracted from wild mature carrot root with no success. In the beginning of the season, i.e., when the wild carrots were still young the methodologies were working smoothly. Now, my positive control keeps on amplifying (suggesting no problem with the PCR reaction) but the new mature root samples do not work.
I have tried:
1. Different extraction methods, from kits to CTAB. Yields are good, 260/280 ratios as measured with nandrop are low (1.7 to 1.4), but not different from the positive control.
2. Adding Polyvinylpyrrolidone (PVP) to the extraction buffer, because I read that mature carrots may have high levels of phenolic and other compounds that can inhibit PCR. No significant differences were observed on yield or 260/280 ratios. PCR keeps on not working.
3. Lower annealing temperature, increased Mg++ content, made touchdown PCR, etc. The positive works, the others not. Again concluded that the problem is not with the PCR.
Currently, I have no idea what more I can do. I am convinced that the problem is in the DNA template, but I have run out of ideas on how to solve it.
Can anyone help? Thank you