Hi, I have been studying human microbiome recently. I am going to do 16s rRNA metagenomic sequencing. Because the concentration of microbial DNA in collected human DNA sample is low, I need to increase the DNA concentration. The 16s rRNA sequencing is targeted to microbial DNA that are mixed with human DNA sample. Because I can not increase this microbial DNA concentration separately, I will increase the total DNA concentration based on human DNA.
I was told to use Speedvac or 2-butanol, but other expert said that plasmid DNA concentration can be increased to Speedvac enough, but gDNA cannot be increased to Speedvac. Do you have any experience with this ? Also, I wonder if the use of 2-butanol does not cause a lot of DNA loss, and if it affects microbial DNA or disturbs sequencing.
Thank you.
I strongly recommend you to contact with company that you will receive ngs service.
Give them complete details.
Hi,
You can use speedvac only if the DNA/RNA is in the water. If it is the EB buffer (for example), evaporation will lead to the change in the concentrations of the carrying solution.
Anyway, if you have enough volume and you just need to concentrate the sample, I can suggest to use column based concentrators (like Zymo Clean and Concentrator) or the AMPure beads. You can also easily enrich only for DNA or RNA and get rid of the other - just incubate your sample with RNase or DNase for the respective enrichment. For example, in our NGS Core Dnase treatment (made on the column during the RNA isolation) is mandatory requirement before RNA-seq library preparation. From another hand, when we isolate the gDNA, RNase treatment allow to get rid of RNA.
Best,
Michail
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