No PCR product - Environmental samples (Oct/28/2004 )
I have been isolating DNA from various environments, such as from crab eggs and filtered water. When I isolate I get like 15ng/ul of DNA( quite low) but I go ahead with the pcr. I use a supermix with pos and neg controls. I use universal 16S and 18s primers but get only pos results from my pos controls. Does anyone have any solutions?
It sounds like there is something in your extractions that is inhibiting the PCR. Did you extract the positive control or is it someone elses? if it is someone elses, try getting them to extract on or two of your samples and see if they get the same PCR results as you.
Otherwise try checking the 260:280 absorbance ratio, it should be around 1.8 for pure DNA, if it is too low or too high then you have contaminants from different stages of the extraction.
Resuspending of the pellets in TE rather than water can sometimes help as well, this removes ions (and hence also some proteins) that may be inhibiting the Taq.
Anyway, good luck!
You might have humic acids, in your DNA. It'll interfere with the dosing. The humic acid will also interfere with the PCR reaction. Try diluting your DNA 1/100 or 1/1000. IF it's not working, try to purify your DNA with a phenol extraction.