Hello!
I'm extracting genomic DNA from Eucalyptus rhizosphere with PowerSoil Pro kit and the highest yield I'm getting is 3,5 ng/uL max. Also, the quality of DNA is around 1,3, wich is not sufficient for shotgun sequencing.
I need at least 10 ng/uL with 1,8 quality rate.
I've already made changes in the protocol (amount of sample, time of shaking on thermoblock, increasing temperatures, etc) and got nothing different.
Can somebody give me some guidance or tips for better results? Following the protocol, I got less than 1 ng/uL :(
The only thing I cannot test/alterate is due to the fact that my lab doesn't have the horizontal vortex adapter, so I use the thermoblock on maximum speed (4000 rpm).
Thanks, everyone!
How about trying more initial material (soil), in several replicates and pool all samples in the alcohol washing step.
I'm saving this attempt for the last case scenario!
Thank you for your answer!
We have used PowerSoil Pro, and for Step 2 (Homogenize) we use a Mini Bead Beater-8 machine that shakes the bead tubes very aggressively. We set it to "Homogenize" for 1min, and this worked well to lyse cells. We did this for corn stover samples, not rhyzospheres. We saw concentrations varied between 4-70ng/uL, but I don't know the quality of gDNA as we've only extracted and put in the freezer for processing at a later date.
Have you tried any freezing methods to help lyse cells? We extract RNA using liquid nitrogen and mortar/pestle grinding - could you use freezing to help? I have not done this with genomic DNA.
I have had good luck talking the Qiagen's tech support, do they have further suggestions to try?
We also planned to set up several replicate extractions as a worse case scenario like Abhijeet suggested above; we didn't need to do this.
Geat suggestions, Lisa McDougall!
Thank you so much. I'll try to improve the lysing step with the equipment we have at the lab. The freezing tip is also a thing I did not think about.
When I have any updates I'll post it here!
Best regards!
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