I am using QIAamp stool DNA mini kit from QIAGEN -the protocol:stool pathogen detection. However, I've hardly got DNA out of my frozen stool samples (around 4ng/ul) knowing that the kit implies that the yield will be 75-300 ng/ul. I followed the protocol word by word and prepared the buffers as indicated. Moreover, i cultured one of my samples to make sure that the bacteria is present and I got heavy growth on blood agar. Thus, I wonder if any one who used this kit could have any explanation, suggestions or modifications of the protocol.
Note: in the lysis step with ASL buffer samples were incubated at 95 for 5 minutes instead of 70 because I'm looking for Gram positive and candida albicans as well.
That is a good idea Michelle,
Beads have the best DNA extraction efficiency for demanding matrices as feces.
Did you add lysozyme? What species are you looking for?
A very good DNA extraction kit is existing from Mobio- based on a first bead pretreatment step.
I am looking for any bacteria present in stool (Gram positive and negative) plus Candida albicans. I did not use lysozyme , it was not indicated in the kit. The kit i am using - QIAGEN stool DNA mini kit -is not based on bead treatment. I have already ordered 2 extra kits and cannot switch into different kind of kit. so any suggestions?
You could try using the Repeat Beading Beating plus column method outlined by Yu and Morrison, 2004 (http://www.biotechniques.com/BiotechniquesJournal/2004/May/Improved-extraction-of-PCR-quality-community-DNA-from-digesta-and-fecal-samples/biotechniques-116769.html). It utilises the Qiagen kit reagents and columns but also has other lysis buffers and cleaning steps. I have used it and it yields a large amount of good quality DNA.
That is a good idea Michelle,
Beads have the best DNA extraction efficiency for demanding matrices as feces.
Thanks a lot for your replies Mr. Michelle and Miss Beatrix. One thing to add is that the samples i got are for newborns at the first day and one month later ,so it seems that the bacteria present in their stool samples is much less than adults, thus would this protocol work for such samples or do is there other specific things to do with the samples before starting DNA extraction -using the same QIAGEN kit.??
You could concentrate the eluate: instead of 100 e. g. 50 microliter.
Best wishes Bea.
I agree that you can concentrate the eluate as Beatrix Stessl has stated. I have used the RBB+C method with adult human samples (less than 200mg) and I have recovered more than 100ng/ul of DNA from them.
there are over 10 kits for stool on the market, some good some suboptimal. try the PureLink microbiome DNA purification kit, it has robust protocols for many mnicrobiome sample types
meconium is very problematic sample, there is little bacteria there so low yield must be expected. as mentioned earlier bead beating is the best (especially as you target bacteria and yeasts, enzymatic lysis would require several enzymes: lysosyme, lysostaphin, mutanolysin and for fungi litycase). since meconium is dense and viscous you should add 2-3 large beads (2-4 mm in diameter) for enhanced mixing
I found the bead beating method is very good too, but I have some trouble about the inhibitor in the DNA when doing PCR, tried PCR degree BSA but still no luck.
How did you guys remove the inhibition?
Regardless of your DNA extraction, it is absolutely critical to add a pre-digestive enzymatic digestion to sphaeroplast the bacteria to remove the cell wall [which are like Kevlar]. Beat beading helps, but that's it. It helps. We use Metapolyzyme which is composed of 6 hydrolytic enzymes against the cell wall of all bacteria {G+ and G-}, yeast, and fungi. This will only work on your sample if it is suspended in PBS at a pH of 7.5. Otherwise you can use lysozyme. It cheap and it's the LEAST you could do.
Not using any enzymatic digestion {and Proteinase K does not count with bacteria cell wall} is a bad idea. Beader beads help, but beat the heck out of your DNA and render it useless if you plan to do long read sequencing or long range PCR. Check out the Omega Universal metagenomics kit and just include a Metapolyzyme pretreatment.
Scott Tighe Chair ABRF Metagenomics Research Group
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