Plant DNA, Calpain maps?

Chris Kafer ckafer at iastate.edu
Tue Nov 10 14:18:43 EST 1998


On Mon, 9 Nov 1998 19:01:12 +0000, "Oliver S. DePeyer"
<saspeyer at reading.ac.uk> 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? 

See  Klimyuk et al,  Alkali treatment for rapid preparation of plant
material for reliable PCR analysis.  Plant Journal (1993) 3:493-494

In a nutshell, you boil small pieces of leaf (5 sq. mm or about the
size of an A. thal. cotyledon) in some NaOH, neutralize and then PCR
with the leaf residue.  My experience is that under 2 kb is
amplifiable.


Good luck.





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