I am looking for any suggestions on how to extract bacterial DNA from stool samples collected and stored in 95% ethanol. (The extracted DNA will be used for 16S rRNA sequencing)
- Kits used?
- How much stool was used in the extraction? How much starting material?
- Does one remove the ethanol before starting the extraction process or is the stool sample homogenized in the ethanol?
I have tried using the PowerFecal / PowerSoil kits (QIAGEN) but I got very little (if any) DNA and the quality was terrible.
Any suggestions / protocols are welcome :)
Dear Patricia,
some time ago I have done the isolation from frozen stool samples (200 mg aliquots stored at -80°C without ethanol) by use of a bead beater (Magna Lyser, Roche) with 0,1 mm zirconia and 3 mm glass beads and 1,2 ml lysis buffer for homogenization and prelysis and then using the PSP Spin Stool DNA Kit (Invitek) with good results. I cannot predict the effect of ethanol in your sample but don't see the need to store the samples with ethanol. Hope this helps a little. Good luck!
Jakob
What is the ratio of ethanol to sample? if it is really high, then you could centrifuge the samples to pellet any free DNA as well as the bacteria (and particulates). Then do a DNA extraction on the pellet.
Qiagen Stool kits are good. But I find that magnetic bead based kits are better than spin columns for stool samples. You tend to get less inhibitors in the purified DNA if you use magnetic beads. So something like the Nuclisens Magnetic Bead kit from BioMerieux is good. I usually do a 10 fold dilution of the stool sample in PBS before doing the extraction to reduce PCR inhibitino too. But this is for detection of enteric viruses, which you'd expect to find in very high numbers. for 16S, it is a trade off between the possibility of inhibitor carry over and detection of rare species.
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