I use the following lysis buffer for cell lysis: 100 mM HEPES, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP pH 8.0. I am interested in protein after cell lysis. The buffer may be different in other cases. You can vary the salt concentration as per your requirement.

See some enzymes like cellulase and lipases

I use Lysozyme in 10mM TrisHCL (pH-7.0) followed by Proteinase K in 1%SDS also prepared in 10mM TrisHCL (pH-7.0)

HEPES or

Glucose : 50 mM, Tris. Cl (pH 8.0) : 25 mM , EDTA (pH 8.0) : 10 mM can also used

Detergents like SDS, TritonX-100 can be used. The cell lysis buffer can contain tris, glycerol,lysozyme, proteinaseK, NaOH. The link below might help you out.

http://www.piercenet.com/browse.cfm?fldID=5559C287-5056-8A76-4E25-8975D8025374

Lysozyme treatment in 0.01 M Tris-Cl buffer, pH 7.0 is OK, following osmotic stress wold be useful for gram negative bacteria.

The choice of lysis buffer depends upon type of Bacteria whether it is gram positive or Gram negative, due to structural differences in their cellwall. The LYSIS BUFFER containing 50mM Tris pH 8.0, 10% glycerol (for stabilization of the protein and prevention of aggregation), 0.1% Triton X-100 (for prevention of aggregation of hydrophobic and membrane proteins) Detergents chosen for the lysis solution should be specific to the proteins, 100ug/ml lysozyme (its MW is ~15Kd. If studying the induction of a protein of similar MW, you may consider not to add it) can be used. The gram positive cellwall is more sensitive to lysozyme than gram negative bacteria. Again 1mM PMSF and /or more anti-proteases (don't add EDTA, if your protein is his-tagged), DNAse 3U + 2mM MgCl final conc can be added after sonication.

For E coli and small scale expression (1ml cultures) we use the following after a freeze thaw step:

Resuspend in: NPI-10-Tween (taken from the Qiagen Biorobot protocol):

50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 1% v/v Tween 20, adjust pH to 8.0 using NaOH and filter before use. Store at 4°C. This suplemented with: supplemented with 1mg/ml Lysozyme and either 3 units/ml of Benzonase or 400 units/ml of DNAse Type I. Stir on an orbital shker for about 30 minutes.

For larger scale we use: 50 mM Tris pH 7.5, 500 mM NaCl, 30 mM imidazole, 0.2 % Tween supplemented with protease inhibitors and DNAseI and we break open the cells using a cell disrupter.

Hi,

It all depends on the species. Alkaline lysis (NaOH and SDS) is a good option for E. coli, but I could never find an easy way for Rhodococcus. In latter case cell growth in the presence of high cc. ampicillin (600 mg/l for 3 h added to a mid log phase culture) can weaken the cell wall, after which lysozym treatment followed by adding 1 M NaCl and 1-2% SDS results good cell lysis in most of the cases.

if you need to lyse bacteria despite of the detergent or chemical solution you'll need heat...or something better..microwave. You can find some papers about it

A mixture of achromopeptidase, lysozyme and SDS will lyse almost all bacterial cells. Achromopeptidase specifically cleaves the peptide bridges,the lysozyme the glycoside link and SDS generally breaks (solubilises) down the proteins.

It depends on the cell you want to lyse. In general we use a combination of SDS, Nacl and Tris buffer with EDTA and it works with a wide range of baceterial cells. We have tried this buffer on bacterial sample from arctic water as well as from soil.

I have used Triton X-100 for both G+ve and G-ve bacteria and it works fine. It's very simple to prepare and use so may be you can go ahead with this before considering other possible options. Cheers.

Hi,

For E. Coli cells we use 50 mM Tris pH 8, 150 mM NaCl and 0.4 mg/mL lysozyme and freeze thaw works best for us.

 Dear Asif,

You only used Triton x-100 for both G+ and G- bacteria without any other buffer or salts? Only Triton x100 in water? This is very important to my research since most the buffers are not allowed in the following steps.

Thanks

Hi

you can use combined enzyme lysozyme and mutanolysin for any type of bacteria present in the clinical samples for efficient lysis then followed with PCI method. You can also use EtNa method of DNA extraction, 1st solution is EtNa alkaline buffer with 2.25mM EDTA and 240mM NaOH IN 71% EtOH - 450ul to the pellet and heat at 85degree C, then centrifuge and remove supernatant, air dry and add 100ul of suspension soln (0.1mM EDTA+50mM TrisHCL+1%Triton-X-100+0.5% Tween 20. vortex and centrifuge. take supernatant directly for ur application like PCR.

All the best