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[Archie]

Hello,

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!

[akhshik]

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.