Bio-Rad ReadyPrep Protein Extraction Kit (Soluble/Insoluble) - Manual (Page 10)

3. With the sample on ice, sonicate the suspension with
an ultrasonic probe to disrupt the cells and fragment
the genomic DNA. Sonicate the sample using 30 sec
bursts, typically 3–4 times, or until lysis is complete.
Chill the suspension on ice briefly between each
ultrasonic treatment. If necessary, transfer the extract
to a microcentrifuge tube when this step is complete.
Note: Disruption of cells by sonication is dependent on the
cell type. For example, E. coli requires longer sonication
times than animal cells and tissues. Yeast cell disruption
requires even more vigorous sonication The addition of
glass beads or use of a Bead Beater (BioSpec Products) can
greatly improve cell lysis of these sample types.
3a. Optional. Nucleic acid can be degraded by adding
~150 U of Benzonase and 2 µl/ml of 1M MgCl
2
to the
extract and incubating the sample at 4–8°C for 20 min
before proceeding to step 4.
4. Centrifuge the tube at maximum speed in a
microcentrifuge (~16,000 x g) for 20–30 min at 4°C to
pellet insoluble material and unbroken cells.
5. Remove and transfer the supernatant to a clean tube
on ice.
6. Re-extract the pellet by adding an equivalent volume
of Lysis Buffer to the pellet and sonicating briefly to
disperse the pellet and lyse any unbroken cells.
Repeat the centrifugation as described in step 4.
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