floating of protoplasts

P. Jones BGYPJ at leeds.ac.uk
Thu Nov 7 12:04:10 EST 1996


In article <327DCE40.3939 at mpiz-koeln.mpg.de>,
   Frank Maiwald <maiwald at mpiz-koeln.mpg.de> wrote:
>Hi,
>
>All barley and rice protoplasts that I isolate still contain
>considerable amounts of cell wall material which I must get rid of
>completely. I would like to include a floating step into my protocol.
>Could anybody suggest a good concentration for the (sucrose) cushion for
>the two cell types?
>
>Thanks in advance					Frank

Hi I isolated barley/wheat and tobacco protoplasts for a couple of years and
found the best cusion to be 40% nycodenz (Nycomed) then resuspend to 20 % and 
overlayer with 10% to allow the cells to float up whilst debris goes down.  
The bonus is that nycodenz is biologically inert (useful when studying effect
of sugars on gene expression).

Paul Jones




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