Thanks Julia for putting together such a thorough protocol....

Hi Julia,

Thanks a lot for your response. My samples were collected from scalp, so I guess this may not be one of the high yield sites. Thanks for the suggestion of using the inner nares as a positive control. We thought about running a positive but didn't know better about what would be a good control. Thanks again for helping me out. I really appreciate it.

Hi Ella,

We've had good experience co-extracting bacterial DNA using this protocol (we use the same eluate for both 16S and ITS sequencing). Depending on the skin site, we may or may not see a band on the gel. High yield sites (e.g., inner nares) can have a band. Did you do a positive control? You might run a positive extraction control, like the nares. In our experience, the combination of the lysis buffer + bead beating is sufficient to lyse both bacterial and fungal cells.

Dear Julia,

I tried to run a PCR using 2ul of the microbiome elute (isolated from scalp swab and air control) and V1_27F and V3_534R primers. I also included a water control in this PCR reaction. I only saw a very faint band around 8kb in the scalp sample and both negative controls. Given that the band size is large( I was expecting a smaller size) and the faint signal of the band, I think this band might be just a background band. I am wondering if you normally see a distinct band when you do the 16S rRNA PCR. The PCR was done using the program provided in 16S amplification section in your protocol.

I think the problem for my experiment is that I didn't break up the cells enough to release DNA. According to the protocol, it looks like we are only using the PureLink genomic DNA mini kit for DNA purification (binding DNA to the column and washes with two buffers). The actual cell lysis was done using the MasterPure kit. Since this kit is designed for yeast DNA extraction, do you think it can affect the efficiency on extracting bacterial DNA? I think I will try to lyse the cells longer in the bead beater and see if I can improve the lysis. I will also really appreciate it if you have any suggestions and tips on isolating skin microbiome that I can try in my next experiment.

thanks a lot for you help.

Hi Julia,

Thanks a lot for your prompt reply. This is very helpful. I will try PCR next.

Hi Ella,

Try the high-sensitivity (not broad range) Qubit kit. We find that NanoDrop is not accurate at low concentrations.

Yield varies very significantly by skin site. For example, for the inner nostril, you might expect 50 ng/uL; for a low biomass sites like the forearm or inner elbow you might achieve 0.5 ng/uL. Run your negative control in your PCR (also include a negative control for the PCR only), and see if you get a detectable band with yield--that will be informative as to potential contamination in your collection method.