Hi everyone,
I´ve been struggling with low DNA yields and weird ratio readings extracted from stool samples collected from fish. I am currently using the PowerFecal Pro DNA extraction kit from Qiagen and strictly following their guidelines, but I keep having the same problem.
To address that problem, I tried to decrease the elution buffer (EB) volume to concentrate my samples more, pass the EB twice through the silica membrane, increase the vortexing time, change the vortex machine (Qiagen suggests vortex-genie, but I tried the Precellys as well), wash the stool sample with PBS prior to the extraction process, incubate the tubes at RT to allow the ethanol to evaporate completely, and also soak the silica membrane with EB for a couple of minutes before centrifuging.
None of these fixed my problem.
Could adding an incubation step (65°C/10min) before the beads beat improve efficiency? Since I don´t know if the kit´s lysis buffer contains proteinase K or not,, I wonder if adding to it and then incubating it would improve the DNA yielding.
Would you happen to have any suggestions?
Thank you in advance for all the help
PS: By low yields, I mean ~1ng/ul and weird ratios >2
You could warm up the EB prior to use (55 degrees or so) and let it sit on the membrane for a minute prior to elution. Sometimes adding in an extra wash can help with the odd ratios.
Another thought. How old is your kit? If the wash buffer was left open it may not have enough alcohol in it to keep DNA precipitated on the membrane so you are washing away DNA during washes.
Hi! i obtain DNA from human stool but useing DNeasy blood and tissue kit from QIAGEN. it has K proteinase. i usually get poor yield, but always enought and with GREAT quality(1,8 = 260/280). i normally obtain 60-100 ng/uL.
My extraction protocol is:
- 65°C for 30 minutes (50-100mg stool + ATL buffer)
- adition of 200ul AL + proteinase K 20ul + glass beads 200mg and vortex (horizontal) for 10 minutes
-10 minutes at 56°C
- add alcohol 100%
-continue with protocol (loading spin columns and wash)
-elution with Elution Buffer QIAGEN (50-70uL)
We use this protocol and use the DNA obtained for PCR for fungus!, qPCR, NGS for mycobiome!
Dear Amy Klocko and Cristian Mena thank you so much for your input!. I tried again today with a few adjustments in the protocol: warming up the stool + lysis buffer at 65°C for 10 min before vortexing, and warming the EB prior to the elution step. Ultimately, I got much more DNA (~8ng/uL - 50uL volume), and the quality was also good (1.8). I think there's still room for adjustments, but I wonder if the animal's age has something to do with the low DNA yields I'm getting. The fish I collected the samples from were tiny and around one month old, making me wonder if their gut bacterial community wasn't established yet...
By the way, Cristian Mena , do you add the 100% ethanol to your samples directly? How does that work?
Thank you once again for all your help!
Hi William Furtado
I was wondering how you went with this in the end and if you made any other adjustments? I'm working with a species that's giving me the same issues that you were experiencing... I was going to try warming the sample + lysis buffer and EB tomorrow but was wondering if you'd optimised any further and had any tips.
Thanks :D
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