PCR of bugs in berries

SG Edwards sge1 at york.ac.uk
Wed Jul 10 04:35:41 EST 1996



On 8 Jul 1996, Walter Hill wrote:

> 
> Hi
> 
> We're trying to develop a method to detect pathogenic microbes which
> might be associated with various berries (e.g. straw, rasp, and
> black) by using PCR.  We have found
> that washes from berries have a strong inhibitory effect on PCR and
> would like to know if anyone has any suggestions for removing
> these inhibitors.  We wash the pellets extensively (3-4 washes)
> before lysing the cells.  We have tried ethanol precipitating the DNA
> but this leads to only a very slight improvement.  The procedure we
> are using also includes Chelex so whatever is causing the inhibition
> is not removed by it.  Anybody have any favorite columns or rapid DNA
> prep methods that they would recommend?
> 
> 
Can I first recommend you try PCR of your extracts again but add BLOTTO 
(dried non-fat milk) to your PCR reactions.  See the paper by DeBoer et 
al Nuc Acids Res (1995) 23 2567-2568.  We tried it on root extracts and 
our PCR products went from nothing to strong bands.  The effect is very 
impressive and we even get bettter amplification of clean plasmid DNA.  
If you want to try it you can use the milk fresh and not bother 
with the preservative (NaN3).  We add 1 microL of a 50 mg/mL dried milk 
in water solution in a 50 mircoL PCR reaction.

If you still have problems then you could try Xanthogenate extraction
which is good at removing/inhibiting proteins, it is also cheap! (Jhingan
(1992) Methods in molecular and cellular biology 3 15-22 AND 185-187.  In
the UK there is a new plant DNA extraction kit "nucleon phytopure" 
(Scotlab, UK) which removes carbohydrates.  This may be worth trying if
you can get hold of it. 

Good Luck

Simon Edwards
University of York
UK




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