Plant DNA, Calpain maps?

Frederik Boernke boernke at nospam.ipk-gatersleben.de
Tue Nov 10 12:35:38 EST 1998


Oliver S. DePeyer wrote:
> 
> I've got two disparate requests and I'd be very grateful for any advice
> anybody could shed on either of them. Please email any suggestions to me
> directly (I have trouble accessing the Usenet frequently). I'm at
> saspeyer at reading.ac.uk
> 
> O.K, problem 1: I have lots and lots of tobacco plants, grown in culture
> (i.e in sterile Jars, MSO agar, none of them more than a month old, no
> flowers). I suspect that many of them are transgenic. I screened some by
> PCR after extracting plant DNA with methods such as Qiaeasy but this isn't
> feasible (IMHO) for more than half a dozen plants due to the sheer amount
> of time it takes.  What I'd really like to do is something like crushing a
> leaf into a PCR tube and run a PCR straight from that - a bit like you can
> run PCR from a bacterial colony, or even a mosquito, crushed in a tube. Is
> there any protocol like that available for tobacco?
> 
> Problem 2: I have some protein sequences and I need to screen them for
> protease cleavage sites, particularly Calpain I & II. What program should
> I be using, and which centre could I find it at? (E.g Sanger, NCBI, etc.?)
> 
> Looking forward to your advices!
> 
> Oliver de Peyer
> 


Hi Oliver,

why do you screen your plants by PCR? This only tells you they have got
the transgene but not whether they are expressing it. All the kanamycin
resistant plants should be transgenic but it doesn't mean all of them
are
expressors. The easiest way of screening in tissue culture is doing 
westerns (if you have an antibody) this only needs small amounts of 
tissue for preparing protein extracts. We usually extract RNA out of our
putative transgenics shortly after transfer of the plants into the 
greenhouse with subsequent northern analysis. This at least tells you 
whether there is RNA (in case of heterologous overexpression) or
supression of the endogenous signal (in case of
antisense/cosuppression).
With our protocol we can easily screen about 30 plants a day. It is
quiete some work but we think it is the best way.

hope this helps
Ricky 

******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de



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