A Procedure for Bacterial Harvesting and Mechanical Lysis

Take a culture of Staphylococcus aureus, a bacterium with a thick peptidoglycan cell wall.

Dilute the culture in fresh media containing antibiotics and incubate it at a warm temperature with shaking to promote bacterial growth.

Transfer the culture to a tube and centrifuge it to pellet the bacteria.

Discard the supernatant and place the pellet on ice to protect the bacteria from damage.

Resuspend the pellet in lysis buffer and transfer it to a tube containing silica beads.

Insert the tube into a mechanical cell lysis instrument and initiate agitation.

Rapid collisions between the bacteria and beads generate shear forces. This lyses the bacteria, releasing DNA, RNA, and RNA-protein complexes.

Ions in the lysis buffer stabilize RNA structures, while reducing agents in the buffer inactivate RNA-degrading enzymes.

Now, centrifuge the mixture again to pellet the debris.

Collect the supernatant containing nucleic acids and proteins for further analysis.

Grow one colony of strains carrying either pCN51 P3 MS2 sRNA or pCN51 P3 MS2 plasmids in three milliliters of BHI medium supplemented with 10 micrograms per microliter erythromycin in duplicates.

Dilute each overnight culture in 50 milliliters of fr