Krumlauf, R. and G. A. Marzluf,
A convenient method for the isolation of crude nuclear pellets.

From Neurospora Newsletter 25:15

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                
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

       DNA concentrations were measured by the diphenylamine method
       (Giles et al. 1965, Nature 206:93)

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