Dear Cynthia,
We have used the method (with minor modifications) described by (Pilcher,
K. E., et al., 2007. Nat Protoc. 2, 1325-8.). Paper describes the DNA
isolation method; you can discontinue the process once you have seperatd
the nuclei.
Briefly, protocol is as follows and is suitable for Axenic as well as
bacterially grown cells.
1)Collect Dictyostelium cells, wash with KK2 buffer and suspend the cells
in cold Nuclei buffer (25mM tris-HCL, 5mM Magnesium acetate, 0.5 mM EDTA,
5% sucrose, pH 7.6). The cell suspension should not be very dense.
2)Add NP-40 (final concentration 2%) and incubate the cells on ice until
complete lysis occurs. Cell lysis can occur in 10 to 15 minutes or even
laser time, check microscopically for clear solution.
3)Centrifuge the suspension at 12,000g at 4°C for 10 min., pellet contains
the nuclei.
Good luck
Santosh
IISc, Bangalore, India

> Dear Dicty researchers,
>
> Does anyone have a good protocol for isolating nuclei from Dicty? I no
> longer have a French press available to me, so I was thinking that we
> could
> try the membrane/syringe method that Roop suggested for cell lysis.
>
> Thanks,
>
> Cynthia
>
>
> Cynthia K. Damer
> Associate Professor
> Biology Department
> Brooks Hall Room 229
> Central Michigan University
> Mount Pleasant, MI 48859
> (989) 774-3455
>
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