crude bacterial DNA prep

Trond Erik Vee Aune trondaun at FJERNDETTE.nt.ntnu.no
Tue Aug 9 02:01:28 EST 2005


Michael Sullivan wrote:

> I want to use PCR to confirm the presence of a large, low copy number  
> plasmid from a bacterial strain.
> 
> Can one simply use a small amount of culture directly in a PCR  
> reaction, or should one do some sort of DNA prep, even a crude one,  
> prior to PCR? If the latter, does anybody have any suggestions for an  
> easy prep to make PCR-quality DNA.

I've done this many times with very low copy number plasmids. I scoop up 
some colony material, boil it for 10 minutes in 10 ul dH20, spin down 
the cell material and use the supernatant as template for the PCR. You 
might have to optimize the amount of supernatant used in the PCR.

What I do prefer to do is to use liquid culture instead of colonies. 5 
ul liquid culture is then boiled, centrifuged and the supernatant is 
again used as template in the PCR. I prefer this method since it's 
easier to get the exact same amount of template in each reaction 
compared to trying to scoop up the same amount of colony material.

Either way I've found that fresh colonies/culture is important for a 
good result.

Trond Erik

-- 
Trond Erik Vee Aune
Department of Biotechnology, NTNU
http://www.biotech.ntnu.no/molgen

- Must be sad being a dyslectic, agnostic insomniac, lying
   awake during the night, wondering if there really is a dog


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