Polymerase Chain Reaction of Agrobacterium tumefaciens

Melissa Rau raum at bio104.alleg.edu
Mon Feb 12 15:38:12 EST 1996

	Unlike every other known dicot, the aquatic plant Hippuris  
vulgaris does not form a gall (tumor) when it has been inoculated with the  
plant bacteria, Agrobacterium tumefaciens. Therefore, to prove that  
Hippusis vulgaris has actually been infected (since no visible gall  
exits), I am trying to analyze its genomic DNA to see if it contains the  
T-DNA region (the transferred DNA region) of Agrobacterium tumefaciens.  
[The T-DNA is the portion of the Agrobacterium tumefaciens plasmid which  
incorporates itself into the plant's DNA following inoculation.] To detect  
this T-DNA region in the infected plant's DNA, I am using the technique  
known as Polymerase CHain Reaction (PCR). 
	I am using PCR to amplify a small region of the infected plants'  
DNA using primers that ONLY match up with the T-DNA (transferred) region  
of Agrobacterium tumefaciens. Therefore, amplication should occurr ONLY if  
the T-DNA region is present (no amplification should occur if it is  
absent) So, IN THEORY, this should be an excellent way to determine  
whether or not Hippuris vulgaris has actually been infected by  
Agrobacterium tumefaciens-- based on the presence or absence of the T-DNA.  
	HOWEVER, I am having some trouble carrying out this procedure. For  
some reason, following PCR (amplification), most of my gel lanes contain  
long "fuzzy" streaks instead of nice sharp bands (which would represent  
the small region of the T-DNA which was amplified ). I assume this might  
be due to non-specific binding of the primers, but I'm not sure.
	If anyone has any experience running PCR with plants or has some  
knowledge of molecular techniques; please write back with some advice!!!!  
				Kristen Cunningham

p.s. I am writing this from someone else's account so please email your  
responses to:  cunnink at alleg.edu

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