Re: 16SRNA sequencing to determine differences between bacteria species

I concur with Derek if you can you probably want to try to grow the
bacteria out in pure culture before you try to amplify the 16s
region.

Right now I'm working with a endophytic plant bacteria that is
extremely hard to culture I ran a 16s PCR on the tissue and needless
to say I had quite a few bands on the gel. From my gel it does seem
there is a band in the same region as the control. I'm planning to cut
it out and having it sequenced. Long story short not everything is
culturable and it maybe neat to also determine if things are present
that you can't readily culture.

On Dec 2, 3:25 am, Derek <dere...@gmail.com> wrote:
> up to you. If you're not trying to conserve plates I'd do a swab per
> plate just to keep them isolated.
>
> Pretty close to a lab protocol at Washington U that a bit of google-fu
> turned up, btw:www.nslc.wustl.edu/elgin/genomics/bio3055/idunknbacteria06.pdf
>
> --Derek
>
> On Dec 1, 10:57 pm, Koz <kozovsk...@gmail.com> wrote:
>
>
>
>
>
>
>
> > Thanks for the link up Nathan, some super useful info there.
>
> > Derek to your point,
> > I will start with some 20 or so TSA plates and see what grows per each
> > swab and then attempt to isolate.
> > Any suggestions on culturing? Should I split each petri in 4?
>
> > Alexander
>
> > On Dec 2, 1:22 am, Derek <dere...@gmail.com> wrote:
>
> > > very true if trying to identify a pure colony- cheap and easy. If
> > > instead you are trying to identify species distribution from a swab
> > > then my understanding is that the pcr step is largely the same but
> > > that you'll have such a mix of species that you can no longer depend
> > > on a simple sanger sequencing. I believe multi-tag pyrosequencing in a
> > > 454 run is the current preferred approach in determining species
> > > distribution, but at $4k a run that's definitely not what you're
> > > looking for.
>
> > > If I'm wrong and there's a simpler way to do species distribution from
> > > swab samples please let me know!
>
> > > Otherwise, you may be best off in attempting to isolate colonies
> > > inoculated from your swabs onto petri dishes and sanger sequencing
> > > individual colonies at $10 a pop or so. That won't give you
> > > distributions but will at least distinctly identify some individual
> > > bacteria.
>
> > > --Derek
>
> > > On Dec 1, 8:16 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
>
> > > > (Specifically Patrik's response):
> > > > On Oct 6, 12:00 am, Nathan McCorkle <nmz...@gmail.com> wrote:
>
> > > > > I was thinking that colony PCR is really quite easy, are there
> > > > > universal primers for 16S? I could get some in the mail in a few days
> > > > > for not more than $5-$15... not sure if I really need that level
> > > > > though, since I think this Prof is a "very very fair" grader.
>
> > > > JGI typically uses universal primers 27F/1391R for bacterial 16S rRNA:http://my.jgi.doe.gov/general/protocols/SOP_16S18S_rRNA_PCR_Library_C...
> > > > The Wikipedia page actually has a table with universal primers as
> > > > well:http://en.wikipedia.org/wiki/16S_ribosomal_RNA#Universal_Primers
> > > > 16S is such a common target in microbiology, that chances are somebody
> > > > at your lab may have some primers available that you could use. Ask
> > > > around among the grad students and postdocs...
>
> > > > On Thu, Dec 1, 2011 at 11:15 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> > > > > See this recent thread, you'll need lab access and tools to perform PCR:
>
> > > > > How to perform bacterial ID (I am trying to determine a Bacillus species)
> > > > >http://groups.google.com/group/diybio/browse_thread/thread/449334a66c...
>
> > > > > On Thu, Dec 1, 2011 at 9:58 PM, Koz <kozovsk...@gmail.com> wrote:
> > > > >> Hi ,
>
> > > > >> I want to do a fairly simple test and determine specific species of
> > > > >> bacteria that exist on different surfaces.
> > > > >> The agar plate/phenotype test does not give me enough precise data, so
> > > > >> I was wondering if there is a relatively inexpensive way to sequence
> > > > >> microorganisms from a collection of swabs?
> > > > >> Pretty new to this and have very limited access to basic lab equipment
> > > > >> so supposedly I would need to send this out.
> > > > >> Any suggestions?
>
> > > > >> Alexander
>
> > > > >> --
> > > > >> You received this message because you are subscribed to the Google Groups "DIYbio" group.
> > > > >> To post to this group, send email to diybio@googlegroups.com.
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>
> > > > > --
> > > > > Nathan McCorkle
> > > > > Rochester Institute of Technology
> > > > > College of Science, Biotechnology/Bioinformatics
>
> > > > --
> > > > Nathan McCorkle
> > > > Rochester Institute of Technology
> > > > College of Science, Biotechnology/Bioinformatics

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