Mixed Culture 16s rRNA Analysis Q&A

[HOMO]

I have a mixed culture plate and I want to do 16s rRNA analysis on it and I have a few questions.

First of all there are no visible colonies, but I know bacteria is growing. Do I just take a small chunk of the medium and do PCR on it with universal primers (I have a procedure for this step)?

Second, because it is mixed culture, won't the PCR be an amplification of a bunch of different 16s from the different bacteria? How do I separate the strains? I have an idea how to do this. After PCR on the universal primers, I can run it out on a gel and excise the separate fragments, then purify so that I have pure samples of all the amplified RNA?

Third, after I purify the RNA, I send it to get sequenced and then I use some database such as BLAST to see what I have?

 

[Ygggdrasil]

Second, because it is mixed culture, won't the PCR be an amplification of a bunch of different 16s from the different bacteria? How do I separate the strains? I have an idea how to do this. After PCR on the universal primers, I can run it out on a gel and excise the separate fragments, then purify so that I have pure samples of all the amplified RNA?

The different amplicons will likely be the same size, so you probably wouldn't be able separate them on a gel. Instead, I would either perform TOPO cloning on the PCR products to create plasmids that can be transformed into bacteria, then isolate single colonies for plasmid purification and sequencing. Alternatively, you could use the PCR products to generate a library for next-generation sequencing.

Third, after I purify the RNA, I send it to get sequenced and then I use some database such as BLAST to see what I have?

Blast would work. People in the field of metagenomics may have developed better tools specifically for mapping 16S rRNA sequences.