I use freeze thaw lysis method for lysis of E.coli and use it for isolating ribosomes. Although the cell lysis happens and i get respectable yields, still I feel the cells lysis is not happening with proper efficiency and a lot of cells go into waste. I cannot use denaturing conditions and cannot include detergents in the prep. Any tips to improve the lysis by freeze thaw method.
The protocol i follow is as follows :
Step 1: Resuspend a gram of cells in 1-2 ml buffer and incubate with 1mg/ml lysozyme with 0.5 mM EDTA in ice for 1 hour.
Step 2: Freeze the cells using liquid nitrogen and that in water bath set at 37 degrees C.
Repeat step 2, 5-8 times
Step 3: Add some DNase and incubate at RT for 10-15 minutes and proceed to clarify lysate by centrifugation.
Can you add a physical disruption method, such as sonication, French Press, or bead beater?
Hi, Adam...French press is a method of choice and i use it when the sample volume is above 3-5 ml.But the volume i discussed for freeze thaw is less than 1ml. Secondly we do not have a bead beater...so we have to resort to the freeze thaw method.Lastly as i am, trying to isolate ribosomes so sonication is not a method of choice here. I am wondering if a reagent like the B-per lysis buffer or cell lytic solution B from sigma can be used for this.
Thanks
(have made some edits in the question for more clarity)
Considering the ease and low cost of growing E. coli, I would not worry too much about maximizing the yield of cell disruption. If you are preparing ribosomes, it is more important that the method be gentle, as opposed to efficient.
Adam, Do you have a view on the use of cell lytic solution supplied by sigma or any similar reagent that uses mild detergents for lysis.?The reagent is a mixture of mild zwitterionic detergents.
I have used the reagent and it does significantly improve the lysis efficiency and the ribosome yields as well. Having said detergents, these may or may not effect the ribosome composition or activity(which is to be tested of course).
I haven't worked with these detergent-based lysis solutions. I prefer to use the French Press.
Late update...but I used the Cell lyticB solution at 0..5x conc. along with the Freeze thaw method...and works very well. We also did not see any obvious aberrations in the polysome profiles.
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