Hello !
I would like to extract gDNA for soil to do NGS. We have the QIAGEN Powersoil kit. I did it twice now. First, according the kit instruction : concentration = 10 ng/uL, 260/280 = 1.4, 260/230 = 0.7. And the second time, I changed the lysis step by vortexing for a longer period and I heated the elution buffer at 50C as ssen on other discussion but my results are still not that good : concentration = 10 ng/uL, 260/280 = 1.8, 260/230 = 0.8.
Do you maybe have an idea on how I can improve my ration to send the sample for NGS?
Thank you !!
The 260/280 and 260/230 ratios are indicators of the quality of your DNA, but if they are lower than 1.8 it does not mean that your DNA is not suitable for PCR amplification or sequencing. I suggest to perform a PCR with bacteria or fungal universal primers to check if there are PCR inhibitors. If the PCR works you should be fine as the quality of the DNA is sufficient enough for downstream procedure.
If the PCR does not work try to dilute your DNA (e.g 1:10 or 1:20 or more) and then test it again via PCR. I had ratios similar to yours but after diluting the DNA template 1:20 my PCR worked fine.
You can also add beta-mercaptoethanol 1% or Polyvinylpyrrolidone (PVP) to the lysis buffer to remove polysaccharides and polyphenols. Use the lysis buffer together with bead-beating procedure.
The instruction compares its best performance with other kits, yielding 60 ng/ul from 250 mg of top soil. How the amount of soil did you prep?
Why don't you, after trying many times, collect all the samples in one tube and do PCI method for gDNA prep?
The 260/280 and 260/230 ratios are indicators of the quality of your DNA, but if they are lower than 1.8 it does not mean that your DNA is not suitable for PCR amplification or sequencing. I suggest to perform a PCR with bacteria or fungal universal primers to check if there are PCR inhibitors. If the PCR works you should be fine as the quality of the DNA is sufficient enough for downstream procedure.
If the PCR does not work try to dilute your DNA (e.g 1:10 or 1:20 or more) and then test it again via PCR. I had ratios similar to yours but after diluting the DNA template 1:20 my PCR worked fine.
You can also add beta-mercaptoethanol 1% or Polyvinylpyrrolidone (PVP) to the lysis buffer to remove polysaccharides and polyphenols. Use the lysis buffer together with bead-beating procedure.
I got good DNA.
As more number samples time would have definitely more what they mentioned (by the time of removing/adding solutions/aliquoting/labeling etc. etc.). Attached the photos.
Do not much bother about the ratios.
I always estimate the quality of DNA by seeing it on the gel and trial PCR not by ratios.
As mentioned by Sang-Min collect the DNA if you want more quantity either reprecipitate or for normal PCRs you need dilutions only.
Good Luck!!
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