Protocol for identification of unknown bacteria

Scott J. Coutts scott.coutts at
Fri Mar 14 20:00:41 EST 2003

Des O'Connor wrote:
> HI Trond
> Im no expert in this area of molecular biology  but isnt  16S sequencing
> done on ribosommal RNA usually after density gradient centrifugation thus
> automatically dispensing withe presence of the microbial  chromosome.

Nah, you dont need to do any of that. Bacteria are easy. As long as you 
have specific 16S primers, all you have to do is a colony PCR with a 
high-fidelity enzyme. Use pfu or kod or something like that rather than 
taq so that you dont get as many errors in the product. Just pick a 
colony, resuspend it in an eppy with 50uL of ultrapure (milli-q etc, or 
some other ultrafiltered) water and boil it for 10 minutes. Spin it in a 
  microfuge as fast as it will go for 10 mins (to remove insoluble cell 
debris) and use a few microlitres of the supernatant (might need to try 
a few different amounts, but probably 2uL will be fine) as template in a 
50uL PCR reaction with your 16S primers.

Once you take a look at a gel and confirm that you have lots of PCR 
product there, do a cleanup using a commercial kit (qiagen, eppendorf 
etc). If you dont have a kit to do it then do a standard ethanol-acetate 
precipitation for short products, resuspend in dH2O and do a PEG 
precipitation. Once you've done that, set up four sequencing reactions, 
two using the 'forward' 16S primer and two using your 'reverse' 16S primer.

... wait for the results, and you'll have beautiful 16S sequence that 
should overlap in middle (as long as you get reasonable runs from 
wherever you get your sequencing done). If they dont, then you'll have 
to do some primer walking to fill in the gaps.

If you need any protocols for anything above, just let me know.

PS: You can buy read-made specific 16S primers from lots of molecular 
biology companies, by the way... just in case you were planning on 
designing them yourself.

PPS: Some people do their colony PCR by simply adding the bugs directly 
to their PCR reaction, and setting an extra 5 minute 95oC hold at the 
start of the program. But I think that is more unreliable, especially if 
you accidently use too much starting material.

> I dont want to be a ''party pooper'' but Im pretty certain if your starting
> from scratch and dont have the correct tech ( ribotyping sequencer etc) then
> I suspect you have some months of hard work ahead and a large bill at the
> end of it.

Nah, you can do it with standard equipment. If you can precipitate small 
DNA molecules (PCR products ~2-4kb) and you have access to a PCR machine 
and sequencer, then there's no problems!

> On a more positive note if you wish to email me privately Ill run the bug
> through our diagnostic lab (gratis) for you if that would help.

Send it, Trond... send it! If you do, will you let us know the results?

Also, can I ask if this is part of a research project or something? Is 
it commercial? Or just out of interest? If you dont want to say, that's 

> "Trond Erik Vee Aune" <trondaun at>
> wrote in message
> news:3E71F004.8000008 at
>>I want to know how to do 16s sequencing on an unknown gram negative
>>bacteria. Preferably I would like to do the sequencing reaction directly
>>on colonies without the need to isolate genomic DNA first. I would also
>>like to know the sequence of the "universal" primers used. Is it enough
>>with only one set of primers to get enough sequence to correctly
>>identify the bacteria?
>>Trond Erik Vee Aune

Scott J. Coutts
  Bacterial Pathogenesis Research Group		
  Department of Microbiology			Ph  [+61 3 9905 4838]
  PO Box 53					Fax [+61 3 9905 4811]	
  Monash University, 3800, Australia

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