Arabidopsis DNA analysis

Christiane Hassel chassel at indiana.edu
Tue Sep 11 12:54:03 EDT 2007


Hi Flow Community,

  I have an investigator who wishes to do DNA analysis of /Arabidopsis 
thaliana/ wild type and mutants.  I was wondering what the best way of 
doing the analysis would be.  I realize there are several investigators 
out in the flow community who have done DNA analysis with /Arabidopsis/, 
so my question to you is, how do you set up a 2-parameter plot for 
selecting the /Arabidopsis /nuclei?  We've done DNA content in 
/Drosophila /and various mammalian cells, and, I understand the usual 
way is FL2-W vs FL2-A linear, but in this case, it would seem we would 
have to crank our voltages up much too high and still not see anything.  
I understand the DNA content itself is very small, and was therefore 
wondering if a log histogram and/or 2-paramater plot are suggested?  We 
prefer to use PI staining as the method, but quoting from Current 
Protocols in Cytometry in cases of small DNA content analysis "the 
contribution of chlorophyll autofluorescence can overlap PI-induced 
nuclear fluorescence on one-dimensional histograms".  Is DAPI the better 
stain in this case?  And, is there a "best" protocol (buffer/chopping 
method) for /Arabidopsis /itself. 

  We have access to a Blue-Red laser FACSCalibur, Blue-Red-Violet laser 
LSRII and a Blue-Red-Violet-UV laser FACSAria. 
  Apologies if my inquiry is too vague or if I've missed this 
information already on the list.  However, any advice, pointing in the 
right direction is greatly appreciated. 

Thanks very much in advance,

Christiane

Christiane Hassel
IU Bloomington
Flow Cytometry Core Facility
Jordan Hall 029
http://facs.bio.indiana.edu/
812-855-7101
chassel at indiana.edu





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