To avoid such a problem, one has to spike environmental samples with targeted DNA and carry out optimization.
Once you spike your desired sample with pure extracted DNA there should not be any problems.
Though there could be PCR inhibitors in the crude environmental samples.
Hi
take a look at this paper
http://www.ncbi.nlm.nih.gov/pubmed/23649952
best regards
Hi,
Most probable answer come from availability of your target DNA when mixed with other molecules. When mixed, primers theoretically take more time to anneal with the proper complementary sequence additionally to sample complexity and inhibitors present in complex samples.
All the best,
Alfonso
DNA preparaion is most crucial in preparing environmental or food samples. We had very good results by using invitek spin column prepared DNA. You can get rid of much inhibitors if you prepare DNA from bacteria grown on plates (even mixed cultures) than from liquid samples.
I work with wild potato species and sometime I have the same problem. I think that is because of high content in plants phenolic and pigments staffs. For example, DNA from Solanum bulbocastanum (purple colored species) in PCR is usually more difficult. To mark up the DNA quality, we take the lives only from yang plantlets or (that is the best) from in vitro plants
Hi, environmental samples are sometimes a bit tricky. I extracted DNA from activated sludge and found that it was necessary to perform the washing step (depends on the DNA extraction kit you are using) for three times instead of only one to get rid of PCR inhibitors.
Lower (often unpredictable) target DNA or gene concentration, inhibitors, DNase, RNase. In the absence of a good column DNA extraction kit, inhibitors can often be diluted out without significantly affecting detection of the target DNA
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