I extract DNA from various sludge samples according to the method by Griffiths et.al (2000). By now I did not have any problem, even in case of sludges which contain a great amount of silage and humic acid. However, all samples which I treated by now were either from a digester running in a continuous mode, or from a batch process, which was running for no longer than 3 months. This time, I have been running a fed-batch process for the last 6 months. I have faced difficulties in obtaining pure DNA samples for the last sludge samples. I am assuming that the biomass concentration and substrate accumulation increased over time, which led to high absorption values at 230 nm. I have tried to modify the DNA extraction method in several ways, however, the only modification which led to pure DNA samples was to decrease the sample mass and to increase the volumes of CTAB solution, phosphate buffer, phenol, chloroform, and isoamyl-alcohol, i.e. to increase the concentration gradients. Even diluting the samples did not lead to better results. The only thing I am concerned about is what happens with the total DNA when the volume ratios of used solvents and samples are shifted.
I think for phenol, chloroform method, if you increase the volume of phenol it won't affect the yield of DNA from your sample. So if you get better result using more phenol, chloroform just go for it. To make sure whether the yield is same, you can use a fluorometer which man specifically measure DNA. Nanodrop is not a good method for quantify DNA amount in contaminated samples because contamination can modify absorbance and give you wrong readings. So try to use both method. Use nanodrop for 260/230 ratio and for DNA concentration use fluorometer.
HI, Stanislava Mlinar
One other option, with may be more expensive and laborious, is to perform a few diluted DNA extraction as you said you did (and find good results) for each sample and then sum them in a single sample. Then, you can speedVac it to concentrate and see If it helps you. You can do it stepwise (1+1, 1+2, 1+3, 1+4, etc) and see how much will still give you good results.
Just a thought.
Wish you all the best on your experiments.
- Badges
- Science method