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, wont 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?
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, wont 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?
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