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DIAGENODE
BIORUPTOR
®
PLUS
USER MANUAL
Diagenode Inc. North America
/ Phone: +1 862 209-4680 // Fax: +1 862 209-4681 // Mail: orders.na
@
diagenode.com
Bacterial Cell Disruption
For cell lysis, we highly recommend using 1.5 ml TPX microtubes (Cat. No. M-50050) or 10 ml tubes (Cat. No. AS-100)
and the corresponding tube holders (Cat. No. UCD-pack 1.5 and UCD-pack10). To guarantee homogeneity of sonica-
tion, the tube holders should always be completely filled with tubes.
Operating conditions:
Tubes:
1.5 ml TPX microtubes or 10 ml tubes
Tube holder:
1.5 ml tube holder (Cat. No. UCD-pack 1.5) or 10 ml tubes holder (Cat. No. UCD-pack 10) with reflecting
bar
Sample volume:
300 μl for 1.5 ml TPX microtubes
2 ml for 10 ml tubes
Sonication buffer:
PBS with protease inhibitor cocktail
Temperature:
Maintain at 4°C by using the Bioruptor® Water Cooler (Cat. No. BioAcc-Cool) or by using crushed ice
Power setting:
H position (High)
Sonication cycle:
30 sec ON, 30 sec OFF
Total sonication time:
10 min for UCD200/300
15 min for Bioruptor XL
Note:
Please note that additional optimization might be required depending on the bacterial strain and growth phase.
Gram-positive bacteria are more resistant to sonication than Gram-negative bacteria because of the rigid cell wall. Cells
in log phase are less resistant than cells in stationary phase. In order to preserve protein structure and activity, avoid a
long sonication.
Protocol:
1.
Collect cells by centrifugation at 1000 g for 10 min at 4°C
2.
Wash twice with cold PBS.
3.
Resuspend cells in cold PBS to OD600 3.0.
4.
Transfer cell suspension to sonication tubes. For optimal efficiency, use the recommended sample volume.
5.
Sonicate at High Power for 10 min (UCD200/300) or 15 min (Bioruptor
®
XL)
6.
Centrifuge at 15.000 rpm for 15 min at 4°C.
7.
Separate the soluble fraction (supernatant) from the insoluble (pellet).
8.
The pellet can be used for extraction of insoluble proteins with a denaturating buffer of choice.
