We are having troubles with DNA isolation from sewadge sludge- we tried isolation with QIAGEN soil/faecal and MP soil DNA isolation Kits. We also modified MP kit and we used phenol-chloroform-isoamyl alcohol modifications for the first steps - still no results. Do you have any advice what to do? We are considering hot phenol method, but maybe we are missing sth (picture attached)
What is exactly the trouble you are having? Low DNA cc, or fragmented DNA? What volume do you use? If concentration is the issue, you can just use smaller volume for the final elution step and/or use larger volume of starting material.
First try to nanodrop your samples to get the concentration. Then you can adjust volumes of samples to use in the pcr reaction.
Hellen Koka and Péter Gyarmati thank you for your suggestions; we checked nano drop DNA reaches up to 400 ng/ul, but it is not visible on gel. is the lack of DNA band onn the gel caused by the contamination (the 260/230 is very low and thus we only observe smears) or nano drop results are not really reliable? anyway we will try with PCR on diluted samples. Once again thank you for your help
Elżbieta Mierzejewska-Sinner nanodrop can give you very high background signal for trizol/phenol-chloroform residues, use Qubit or similar
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