Re: Plant ploidy

Dear Sue,

Just a short remark: It is not recommended to mix two samples that were
prepared independently. Instead, I recommend using the internal
standardization procedure in which the nuclei from standard and unknown are
isolated and stained simultaneously.

You may be interested to check our recent paper with a description of
suitable procedures for plant DNA flow cytometry, a list of useful hints and
a troubleshooting guide:
http://www.natureprotocols.com/2007/09/06/estimation_of_nuclear_dna_cont.php


With the best wishes, Jaroslav

**********************************************************
I am pleased to announce publication of the first book on plant flow
cytometry:
http://www.wiley-vch.de/publish/en/books/bySubjectLS00/ISBN3-527-31487-3/?sID=69583a0477e
f2333f7b43b719b83fac7

**********************************************************
Dr. Jaroslav Dolezel
Laboratory of Molecular Cytogenetics and Cytometry
Institute of Experimental Botany
Sokolovska 6
CZ-77200 Olomouc
Czech Republic

Tel.: (+420) 585 205 852
Fax: (+420) 585 205 853
Email: dolezel@ueb.cas.cz
Web: http://www.ueb.cas.cz/Olomouc1/LMCC/lmcc.html
**********************************************************

Visit the first forum and blog on plant flow cytometry:
FORUM: http://flowerdatabase.20.forumer.com/index.php
BLOG: http://flowerdatabase.blogspot.com/

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


----- Original Message ----- 
From: "kathy schell" <kschell@wisc.edu>
To: cyto-inbox
Sent: Friday, March 14, 2008 8:22 PM
Subject: Re: Plant ploidy


> Hi, Sue,
> Our plant folks used this same protocol that Joanne gives-with PI.  A
> couple of other things, however.
> 1.  Being gentle as possible in chopping up the plant tissue very fine can
> be critical in obtaining plant nuclei.  Your investigators should probably
> try some variations (more or less aggressive) on the technique.  It took
> one group here several tries to actually get nuclei. (Amazing what that
> old microscope can tell you.)
> 2.  Find a standard and stick with it. We recommend that they prepare a
> standard, an unknown, and then a mix of the two to get accurate ploidy
> measurements.  All analysis should be compared with a known amount of
> DNA--even if this is a species that they are not usually using (our folks
> use soy beans).
> 3.  We read this in FL2 (better CVs there) and trigger on FL2 only. We
> generally start by setting the standard at channel 400 and then look at a
> lot of the unknowns. If most are above the standard, crank the detector
> down and set it at 200 (or whatever).  If there are some below and above,
> as was found here, 400 was a good place for the standard.  Always run on
> Lo with the tightest core stream you can get.  These are messy.
> Hope this helps.
> Kathy
>>Hi Sue:
>>One of my investigators used the protocol on this website
>>http://www.ueb.cas.cz/Olomouc1/LabDol/Research/Flow_cytometry/Protocols/Two-step_proc.h
t
> m
>>and had very nice histograms with PI. Have your investigator give it a
>>try.
>>Joanne Lannigan
>>
>>On Tue, 11 Mar 2008 17:14:41 +0000
>>  S Newton <S.Newton@sheffield.ac.uk> wrote:
>>>Hello all
>>>
>>>I was hoping for a few words of wisdom regarding looking at plant ploidy
>>>by
>>>flow. I have attached the protocol our user is following, when the
>>>samples come
>>>to me, I cannot get a clean peak. There is so much debris in there that
>>>any G1
>>>peak is either very broad, or masked by the debris, it is also very
>>>difficult
>>>to determine if there are any subsequent peaks. I am using either a
>>>FACSCalibur
>>>or FACSort to look at these samples, and the DNA is stained with PI.
>>>
>>>I have tried thresholding on FSC then on SSC then on FL3 and finally on
>>>FSC and
>>>FL3, but still no joy.
>>>
>>>Any advice greatly appreciated (as ever)
>>>
>>>Sue
>>>--
>>>Sue Newton
>>>Flow Cytometry Technician
>>>DU27. D Floor
>>>Medical School
>>>University of Sheffield
>>>Beech Hill Road
>>>Sheffield
>>>S10 2RX
>>>
>>>Phone number: 0114 271 3023
>
>
> -- 
> --------------------------------------------------------------
> Kathy Schell
> Supervisor, UWCCC Flow Cytometry Facility
> 600 Highland Ave.  K4/535
> Madison, WI 53792
> Voice: 608-263-0313
> e-mail:  kschell@wisc.edu