This procedure describes a convenient method for the isolation of crude nuclear pellets from N. crassa. The method, an adaptation of the one developed by Hautala et al ((1977 J Bact 130:704-713) utilizes a Braun homogenizer to disrupt the cells. The main advantages of the technique are that the cells need not be froen, large amounts of material can be handled, the homogenization is fast and easily controlled, fewer omni-mix steps and shoter times are required to release the nuclei, yields are comparable (75%) to those obtained using the french pressure cell, and lower concentrations of Ficoll will stabilize the nuclei. The crude nuclear pellets are used to prepare DNA and pure nuclei.
Germinating conidia (14 hrs) are harvested by filtration and rinsed. The Braun
homogenizer disrupts cells via high speed shaking (4000 rpm) with glass beads.
Typically 90 g wet weight of cells are used in each isolation. The 90g are distributed
among four 75 ml glass homogenizer bottles. Each bottle contains 50 g acid washed
glass beads (0.45-0.50 mm), 10-15 g cells and 11 ml isolation buffer A
(Hautala et al 1977). The isolation buffer, however, contains only 5% ficoll 400.
The cells are kept cold during the homogenization by a jacket fed with siphoned
CO2. The cells are homogenized in 30 sec pulses followed by 30 sec rests.
Table 1 shows that optimum yields without lysing nuclei are obtained using 90 sec
total homogenization time. The yield at 120 sec is the same but 20% of the nuclei
have lysed. The homogenates plus beads from the four bottles are combined in a
beaker and allowed to settle for two minutes. The homogenate is then decanted
from the beads. The beads are rinsed three or four times with 50ml of isolation
buffer and are saved for reuse. The homegenate and rinses are combined and their
volume adjusted to 300ml . The mixture is then omni-mixed for 10-15 minutes at a
setting of 6.0, The solution is then centrifuged at 700 x g in large plastic
centrifuge bottles for 10 minutes. The decanted supernatant is saved.
The pellet is resuspended with a syringe in isolation buffer the volume adjusted
to 300ml and omni-mixed a second time using the same conditions.
The solution is centrifuged and the second spin is combined with the
first supernatant. The crude nuclear pellet is obtained by centrifuging the
combined supernatants at 9000 x g for 50 minutes. We routinely obtain yields
of 65-75% based on DNA content using this method. See Table 11.
The entire procedure requires about four hours. it is possible to handle 180g
of cells by running two homogenizations. While the first homogenate is omni-mixed
and centrifuging, the second homogenate may be started in the omni-mixer.
By overlapping the centrifuge and omni-mix times in this manner and combining all
the supernatants to spin down the crude nuclear pellet we can handle 180 g
in four hours and 360g conveniently in a day.
(Supported by Grant GM-23367 from the National Institutes of Health).
- - - Department of Biochemistry and The Developmental Biology Program,
- - - Ohio State University, Columbus, Ohio 43210.
TABLE 1
Efficiency of Cell Disruption with the Braun Homogenizer
% yield and distribution of DNA
time (sec) of crude crude
homogenization nuclear membrane nuclear
---------------- pellet pellet supernatant
------ ------ -----------
0 0 100 0
30 25 70 5
60 39 57 4
90 75 20 5
120 73 8 19
150 57 6 37
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homogenizations were performed in 30 sec pulses followed by 30 sec rests
TABLE 11
Yield Comparisons of DNA Using Different Techniques
Method of % yields based on DNA
Homogenization whole cells crude nuclei pure nuclei
---------------- ------------ ------------ -----------
1. french pressure cell 100 70-80 25
(cell frozen)
2. french pressure cell 100 65-72 22
(cell cold)
3. Braun homogenizer 100 65-75 26
90 sec
4. Hand shaking with 100 20 2-3
glass beads 10 min
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DNA concentrations were measured by the diphenylamine method
(Giles et al. 1965, Nature 206:93)
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