single colony PCR

Sean May [NASC] sean_may at thale.life.nottingham.ac.uk
Tue Feb 1 06:40:38 EST 2000


PCR from bacteria sometimes appears to fail because of the amount of
material added to the PCR.
In other words, if you add too much of the colony, then bacterial/substrate
materials may inhibit the PCR.
In my experience this is particularly true of agrobacterium PCRs.

A good piece of multitasking is generated when you pick the test colon(ies)
into broth and set them growing, then extract a small aliquot for template
(e.g. 0.1 ul) after the bacteria have had time to disperse into the medium
and start dividing (e.g. 1 hour). The PCRs that are positive then already
have an overnight started for them :)

Equally well, just touch the colony - you run more of a risk from inhibition
if you pick the whole colony - if you can see it on your pipette tip (or
toothpick) then you may have too much for an efficient PCR.

Godfrey's technique also works well.

I hope this helps,

Sean

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Godfrey <Godfrey.Neutelings at univ-lille1.fr> wrote in message
news:B4B34FA6.407%Godfrey.Neutelings at univ-lille1.fr...
>  dans l'article v04210101b4a54a3574ef@[128.206.80.4], "Peter L. Schuerman"
ý
>  schuermanp at missouri.edu a Ècrit le 14/01/00 23:00 :
>
>  >  Can anyone recommend a good method for single-colony PCR that is more
>  >  elaborate than simply using cells directly in the reaction?  In
>  >  particular, I'd be interested in a method that works for
>  >  Agrobacterium.  In my experience directly adding cells works well for
>  >  E.coli but I have been getting poor amplification and inconsistent
>  >  results when I have tried this with Agrobacterium.  Any help would be
>  >  greatly appreciated.
>  >
>  >  Regards,
>  >  Peter L. Schuerman
>
>
>  Simply pick a single colony and place it in 50 microL of water. Boil for 2
>  min, place on ice. 5 microL is enough for a 50 microL PCR reaction.
>  Works fine
>
>  ---


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