4 replies to this topic

[alkb]

Hello all!

I am new in this forum. Sending sunny greetings from Germany...

I started to work in terrestrial ecology field few weeks ago. My project is to asses microbial diversity in polluted soil using qpcr. But I already faced first problem, which is DNA extraction. I tried with protocol they usually use in the lab for "normal" soil. The concentrations were very law, the most problematic is 260/230 ratio (around 0,7 -1,4). I also tried with NucleoSpin Soil Kit, results pretty the same...

I need ideas, advices... how to isolate relatively pure DNA with reasonable concentrations from soil, enivronmental samples

Thank you in advance.

[gebirgsziege]

have you tried a pcr with your DNA and checked the yields/quality of the result?

Maybe the bad reading comes from something in the polluted soil interfering with the photometric reads. I have the experience that with soil these photometric and quantifications methods are only partly reliable.....too many factors that can influence your results...

[alkb]

gebirgsziege, on May 11 2010, 11:02 AM, said:

have you tried a pcr with your DNA and checked the yields/quality of the result?

Maybe the bad reading comes from something in the polluted soil interfering with the photometric reads. I have the experience that with soil these photometric and quantifications methods are only partly reliable.....too many factors that can influence your results...

Thanks for your answer. No, I didn’t run pcr yet, because I though good dank quality is a "must be"!!!

[gebirgsziege]

It is good if you have, but I had horrible samples which were working perfectly, so I would give it a try.

[Maddie]

I think humic acids have the same absorbance as DNA, so your concentrations might be totally wrong.
You can always add a cleanup step if you want with a Qiaquick or MinElute.
Try the PCR and don't forget the BSA.