This article details a protocol for lysing Staphylococcus aureus to extract nucleic acids and proteins. The method involves mechanical cell lysis using silica beads and subsequent centrifugation to isolate the desired components.
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 fresh BHI medium supplemented with erythromycin to reach an OD 600 of 0.05. Grow the cultures at 37 degrees Celsius with shaking at 180 RPM for six hours. Transfer each culture into a 50 milliliter centrifuge tube and centrifuge the tubes at 2, 900 times G for 15 minutes at four degrees Celsius, then discard the supernatant, freeze and store them at minus 80 degrees Celsius or keep the pellets on ice and directly perform mechanical cell lysis.
To perform mechanical cell lysis, resuspend pellets in five milliliters of buffer A and transfer the resuspended cells to 15 milliliter centrifuge tubes with 3.5 grams of silica beads. Insert the tubes in a mechanical cell lysis instrument and run a cycle of 40 seconds at four meters per second. Centrifuge the tubes at 2,900 times G for 15 minutes, then recover the supernatant and keep it on ice.
Make sure that the supernatant is clear, repeating the centrifugation if it is not.
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