I'm planning to conduct Metagenomic analyses on DNA extracted from soil using the ITS and 16S primers. However, I'm getting bands at 2k or 3k on the gel, even though my ratios and DNA concentration are within the acceptable range.
The 260/280 ratios are approximately 1.9 and 260/230 ratios of approximately 2 and DNA concentration measures around 250-300ng/ul.
I was expecting a thicker band above 10k. I'm concerned about the quality. Should I proceed with the metagenomic analyses or should I extract the DNA again modifying my method? Do I have too much smears? I use Dneasy Pro kit for soil extraction.
Info:
Primers: Crude DNA extracted from soil with Dneasy Qiagen PRO kit
Gel %: 1% gel
Ladder full name: Lambda DNA/HindIII Marker and 1kb plus
Time and Voltage: 30 mins at 80v and 1h at 80v
Sample Volume and concentration: 1 ul of 6x DNA Loading Dye and 5ul of DNA, 1A 1:1:4
Ladder conc.: 1 ul of 6x DNA Loading Dye and 5ul of Ladder and 1:1:4. 1Kb is 1:5
Read my research virus is living and its treatment whole concept is given their
In the past I would use the following kit to extract microbial DNA from the soil: https://www.zymoresearch.com/collections/quick-dna-fecal-soil-microbe-kits. I never had any issues using this kit, and I always had great yields and really good quality (i.e. ideal 260/280 & 260/230 ratios). I never ran these extracts on an agarose gel afterward, but I would suspect there would be some smearing if I did...but I can't say for sure.
In regards to your metagenomic sequencing, I would suggest that you just send your samples to a company experienced in doing this. In my case, I would send my samples to GENEWIZ. They would do the preparation and the metagenomic sequencing. Upon completion, they would send me all the sequence data. I could then analyze the data using my own protocols/applications. This was definitely a much cheaper alternative, plus if you're a first-time user they usually give a discount! :)
Hope this helps a little. Feel free to reach out if you have any further questions...I'll do my best to assist.
The gel picture of DNA extracts looks smearing. It is not very surprising if the sample was bead processed fiercely and a bit long. The spectral parameter looks good.
With this DNA material, you can go further for PCR to get 16S or ITS fragment. The PCR products will used for amplicon sequences. If you would like to do short-gun metagenomic sequences for the genomic DNA, you can send to sequencing company, they will check the quality and quantity of your DNA extracts with good instruments, and contact you whether the samples are good or not for sequencing.
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