Hello Researchers,
I am performing leaf endophyte 16S amplicon from Arabidopsis plants grown in natural soil in growth chamber. I have collected the leaves (5 weeks), surface sterilised and lyophilised the leaves before DNA extraction. After DNA extraction, I've performed 799F-1391R amplification as per protocol by Chen et al. (Chen, T., Nomura, K., Wang, X. et al. A plant genetic network for preventing dysbiosis in the phyllosphere. Nature 580, 653–657 (2020).)
However, I see no bands for microbiome after PCR in agarose gel, although a band for mitochondrial DNA is there. Can anyone guide me what could be the reason?
Details of PCR: Template used 10 ng. Run for 35 cycles. Negative control is fine and positive control (Synthetic community) giving desired band. Is it due to very low biomass of microbiome in samples, How to troubleshoot?
Workaround:
- Try, if possible, qPCR to see if there are any signal, or
- Try to use maximum possible amount/volume of DNA which could increase the concentration of template in reaction
- Try to concentrate the DNA solution, so that you can use more template per unit volume from solution in PCR reaction
- Best would be to use more starting material which could give you more target DNA during DNA extraction.
Dear arijit,
your experimental setup is doing well.. i would suggest that increase the template DNA for PCR as well as you can check the sample sample once by qPCR. If u observe the same result, then u try to optimise the DNA preparation method for the sample.
Another thing just for curiosity u can use the culture based method to make sure and estimate the bacterial load of your sample.
Sounds like the issue is you are looking for a low-abundance template. You'll need more DNA.
What DNA extraction protocol did you use? You might need to add in grinding in liquid nitrogen, freeze-thaw cycles, or a proteinase K digestion.
Hey guys, thanks for the inputs, it is indeed a low biomass problem and increasing starting amt. solved the issues
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