Hello,
I am lysing E.Coli expression strain containing Maltose Binding Protein. I am having trouble in determining the amount of lysis buffer (20mM Tris-HCl pH 7.4 and 0.5 M NaCl) that I should be adding to my cell pellet (in terms of ml of lysis buffer/gram of pellet). I want to keep it minimum so that I do not have to run long runs of subsequent affinity chromatography.
I also sonicate after lysis buffer resuspension of cell. How long should I sonicate (Q500 GE sonicator). The manual suggests 12 mins of total on time and 24 mins of off time. Thats too much for my 4 tubes of 30 ml tubes each? Can i reduce this time or maybe work with higher volumes of cell lysate each batch?
The lysis method we use is to resuspend the pellet from one liter of mid-log-phase culture in 30 ml of lysis buffer (including protease inhibitor cocktain, MgCl2, DNase, and lysozyme), then pass it two or three times through a cold French Press at 18,000 psi. This is much easier to control than sonication and doesn't heat the sample.
By the way, 12 minutes of sonication is way too long, unless you are doing it very gently. Check the suspension under the microscope for intact cells after each 30-second burst to decide when to stop.
A good starting point would be to resuspend your bacterial pellet in 5 ml of resuspension/lysis buffer per 100 ml of culture. Sonication needs to be empirically determined for each sonicator/probe pair. This is quite easy. Put a similar volume of lysis buffer in a tube that you would have if you were lysing cells. Put the probe in the buffer and sonicate while adjusting the power upward to the point the solution is moving but not flying or foaming all over the place. Basically you are trying to find the maximum sonication power you can use without foaming your samples. After that sonicate your samples on that power setting 3 times for 20 seconds each time on ice (alternate between your samples to allow cooling between sonications). The lysate will appear somewhat clearer after sonication than the starting resuspended bacteria. Clear your lysate by centrifugation and proceed with affinity chromatography.
hi,
You can also check the fall in OD at 600nm after sonication treatment with time lag. Initially presence of bacteria will show some OD but as after sonication their is drastic fall in OD.You can optimise it by taking initial rading(before sonication),5 min after sonication and 10 min after sonication,once you get OD near to blank (without bacteria in media) means lysis was done .
Also microscopic observation is also good one to check the lysis step.
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