Hi,
I am using rosetta Ecoli cells for expression of recombinant cells. After optimal time expression, I am keeping the cell pellet at -80 degree celsius. I am resuspending cells in lysis buffer (Tris ph 8, 150mM NaCl, 1mM PMSF) by 5ml pipette. I find that cells stick to each other and is often very difficult to separate them. I usually use PandaPLUS 2000: Laboratory Homogenizer to disrupt cell. So. it becomes very difficult to add such clumps to machine as it will damage the homogeniser. Can anyone suggest any other way/trick to overcome such problem??
Hi Haaris
I have following points for you to consider: -
1.) Your media - There are certain media components which can lead to excessive clumping of bacteria
2.) Are you incubating your bacterial culture in a constant shaker - If not, incubate it in a shaker.
3.) Addition of glycerol or Tween 80 can solve your problem
4.) use an insulin syringe and pass your culture through it 20-30 times.
5.) use filters - first 50 um - then gradually coming down to 5 um.
Hi
how did u collected the bacterial pelllet?
may be the centrifuge rotation was very fast and there for pellet compaction occured
the viscosity of bacterial suspension usually is due to DNA
try sonication with increased time to break DNA large molecules
Best regards
Sonication should do the trick. Keep them on ice while sonicating or sonicate in the cold room. We usually do not add detergent until after sonication to avoid foaming. You can also try to time your prep w/o having to freeze the cells at -80 to avoid lysis/clumping.
Another way to break up the nucleic acids is to add RNase, DNase, and MgCl2.
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