How to clean extracted faecal DNA? (removing PCR inhibitors) - (Jan/26/2012 )
I'm currently finishing a project regarding Real-Time PCR analysis of bacterial DNA in faecal material.
My stool samples come from patients with rather nasty bowel diseases and I extracted DNA with a protocol similar to this one. The method I was using combines heating, mechanical and enzymatic treatment and there are 2 precipitation steps with pure ethanol & later one isopropanol. Furthermore I cleaned the centrifuged pellets 3 times with 70% ethanol after precipitating and before drying and resuspending.
The DNA concentration for the samples is usually between 0.5 and 2 mg/ml and the 260/280 ratio is > 1.8.
260/230 is poor (between 0.7 and 1.8 depending on the sample).
I normally have to dilute my samples to at least 1:10 prior to using them in Real-Time PCR in order to get no inhibition.
When using TaqMan assays I could dilute them and everything was fine but for some bacteria to be detected there are just SYBR Green primers available.
For these I get quite a lot of inhibition (sometimes even when diluted 1:1000).
Is there any way to clean my samples now that they are already extracted?
I'm not sure what kind of inhibitors are in there but the resuspended DNA extracts are sometimes a bit viscous and colourful (yellowish, brown).
I already tried to using BSA in a concentration of 500 ng/mL (I think) and some proteinase K treatment as well - to no avail.
I didn't want to use spin columns (or a commercial kit) in the first place because much DNA is lost when run through them and I need to work quantitatively.
Thank you very much for your answer!
the best way is to run large amount of them on the agarose gel and re collect them this would help to reduce DNA loss.