help: plant nucleus prep
Leonard N. Bloksberg
bloksber at pilot.msu.edu
Sun Jan 2 13:01:00 EST 1994
In Article <2g6v3nINNcha at dns1.NMSU.Edu> "twei at spock (Tao Wei)" says:
> Hello, everybody
> Recently I am preparing nuclei from tomato leaves to get nuclear extracts.
> As a start I followed a protocol from a friend. Leaf tissue is homogenized
> in chilled homogenization buffer (10 mM MES pH 6.8, 10 mM NaCl, 0.25 M
> sucrose, 5 mM EDTA, 0.2 mM PMSF, 0.15 mM spermine, 0.5 mM spermidine, 0.2%
> Triton X-100) (1:3 w/v), filterd through 2 layers and 6 layers of
> Miracloth respectively and then centrifuged at 2000x g for 10 min.
> Precipitate is resuspended in 10 ml of homogenization buffer and
> centrifuged again. Initially this washing step can effectively separate
> nuclei from chloroplasts. After I ran out of the homogenization buffer I
> had to prepare more following the recipe exactly the same as before.
> Unfortunately the newly-made buffer could not allow the separation of
> nuclei from chloroplasts, both nuclei and chloroplasts are precipitated. I
> tried different sucrose concentrations from 0.25, 0.5, 0.75, and 1.0 M,
> different pH from 6.0 to 7.0 and different salt concentrations from 10 to
> 50 mM. Even though I found pH is a factor of the most importance, no
> unique pH can work as well as the homogenization buffer (pH 6.8) used
> initially. It is so strange to me. I do not have any idea about what is
> going on there.
> I also tried to use Percoll step gradient (10%, 30% and 60%, or 30%, 60%
> and 85%) to separate nuclei from chloroplasts. In all these trials,
> chloroplast seems to be very heterogenous in density and distributed among
> all three gradients although most chloroplasts floated on top of percoll.
> I could not see any nuclear band. Could anybody out there have any
> suggestions or working protocols to help me out?
I have never had this problem with tomato nuclei. They always come clean
for me. Since the purpose of the Triton X-100 is to lyse chloroplasts, the
fact that you have any chloroplasts left to co-migrate with your nuclei
suggests a problem with the triton to me. I use 0.5% Triton. Also, check
that your Titon is fresh. I have used ancient bottles with no problem, but
it can't hurt to check. I also try for 1:5 to 1:10 ration in the first
grinding. I assume you are following green color to see chloroplasts.
Good luck, and please drop me a note to let me know what you are doing with
your nuclei. I may want to compare notes on some other things.
. Leonard N. Bloksberg
. bloksber at pilot.msu.edu
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