I have used utensil sponges as the filter in wastewater treatment. But now I wonder how to extract effectively antibiotic resistance gene from these sponge. Could you suggest me some ideas?
It really depends on your purpose. Are you just wanna to get some of those genes or you wanna to detect their abundances in the samples? If you only wanna to get those genes, just do a pre-incubartion to increase the concentrations of the target genes. For instance, if you are interested in tet-resistance genes, you can mix the wastewater with fresh culture media such as LB or R2A and an appropriate concentration of tet. After incubation, just isolate the total genomic DNA using a kit (e.g., MoBio Power Soil DNA Isolation Kit). The resistance genes are in the DNA samples.
If you want to detect the concentration of the target genes in the wastewater sample, do not do any pre-incubation but just isolate the total DNA directly from the samples.
With the DNA, you can use PCR to amplify the genes you want. It should be relatively easy.
Thank you for your answer Mr/Mrs. Zhang. I tend to evaluate the abundances of ARGs in the sponges. However, shaking the sponge cube (1 mm3) in neutral solution to release bacterial cell seem that doesn't work well. it may be that bacterial cells get stuck inside the cube. I haven't solved this problem out.
In this case, it seems that the isolation is not easy. Is the sponges easy to be broken during the bead-beating process?
Nope! the sponges I used is kind of very good quality so it is not easy to be broken.
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