dna qualification

Timothy Paul Bushnell bushnt at cortez.its.rpi.edu
Fri Dec 1 09:16:23 EST 1995


>
>   I am in the process of developing a lab wherein my students will 
>identify the presence and rate of movement of Agrobacterium tumefaciens 
>within dicots, primarily legumes.  I have considered qualitative analysis 
>of viral/bacterial DNA and RNA using ELISA or agarose gel electrophoresis 
>but, because of a limited budget, alternative methods may be needed.  I 
>realize if there were cheaper methods of doing this they would be 
>commonly known and, not having come across any references as such, I am 
>afraid they may not be available.
>
>   We are also quite limited as far as equipment so any help in obtaining 
>protocols on the identification of the presence of DNA/RNA using 
>inexpensive processes would be greatly appreciated.

Jerry,

	Interesting idea!  I have a suggestion that might be useful
and not expensive.  What you'll need is a fluorscent scope with
a rhodimine filter, and a 16S rRNA probe targeted towards
Agrobacterium (If one doesn't exist, it would not be difficult
to design one)

	Using a 16S rRNA targeted probe, you could determine if the
Agrobacterium is activally growing (there is a direct relationship
between 16S rRNA content and growth).  You might have to 
experiment how best to section the material.  I would suggest a
simple paraffin embedding, followed by sectioning.  THen you could
remove the paraffin, mount and fix the section, then probe
with the oligo.

	There are many references out there for using 16S rRNA probes
for detecting bacteria.  I would refer you to:

Braun-Howland, Danielsen and Nierzwicki-Bauer (1992) Biotechniques
13:928-933

	I hope that this helps

Tim Bushnell
Rensselaer Polytechnic Institute
Troy NY 








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