We are looking for a way to process a high number of stool samples. We currently use a Qiagen kit, which is labor intensive and can only extract 12 samples at a time. Do you know of any machines or kits or other things we can use to improve our extraction process? We are looking into devices like the QIAcube, or other automated systems. I would also appreciate any advice you have about purchasing new machines for the lab.
Hi,
the extraction of DNA by feces is a problem, we have changed our "life" since when we use the Qiagen EZ1 advanced XL, with this machine we can extract until 14 sample at a time in fully automatic . The kit is the follow : EZ1 DNA Tissue Kit (Qiagen). we use the card " with bacterial protocol", (time of extraction 15 minutes) but you can use another card with the protocol that you need!!
good work!!!!
You may search for this article: http://www.ncbi.nlm.nih.gov/pubmed/19252449. But as far I know this method is really time consuming but you can get pure total DNA from the samples.
You may see this and use it....................Trujillo, A.A., et al. Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus. J. Clin. Microbiol. 44 (4), 1405-12 (2006
You can use the Automated DNA extraction machine, it can process 48 samples per time.
You can amplify the 16s DNA from the sample directly, try Phire® Animal Tissue Direct PCR Kit.
Thanks. The main problem we have is all of the contaminants in the fecal matter. We have a bunch of steps in our current protocol to remove contaminants that might inhibit PCR and sequencing. Extracting samples by hand as we do now is possible when we are working with 200 samples over an extended period of time, but soon we will have 8,000, which we really cannot do in a reasonable amount of time.
In case of stool samples, the best option would be to use MoBio labs fecal DNA kit, there are various scales depending on your sample number eg: high throughput 96 well format.
This can be performed manually with common instruments in the lab or be fully automated end to end say by co-ordinating with the liquid handler/robotics machine you are currently using.
I hope this helps, esp with regards to your concerns about pCR inhibitors from fecal samples.
A vacuum extraction system in combination with the qiagen kit can decrease the extraction time. Or you might like to go to a 96 well plate system like Magna pure.
nowadays we have a speciec specific primers for DNA extraction from stools.....try use Qiagen kits for DNA extraction from stool
Maybe it is also worth to try it without extraction - just add a very diluted tiny amount and increase only the first denaturation to maybe 10 min at 95 degree. But it might have to be at least in a 1:100 dilution (better 1:10.000). Otherwise it might inhibit the PCR. I think in stool samples you might have heaps of bacterias....that would possibly be the easiest way :)
We need to do a high quality extraction because we usually end up sequencing the gDNA, or doing other molecular tests. Our samples are also preserved in RNAlater before we extract them.
We currently have no automated system or any kind of robotics, as much as I would like one. We do all of the pipetting manually.
Thanks for your suggestions, I will take a look at some of them and see if they fit with our lab.
Hi Elizabeth,
This is a bit late, but just in case you haven't found it yet:
The HMP consortium members (and several other labs) have use the powerSoil kit from MoBio for fecal samples with excellent results.
It has a 96-well format that does not require robots. You'll probably need access to a tissue-lyzer, though.
best,
Chris
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