Adding TurboNuclease (http://www.accelagen.com/TurboNuclease.htm) to the lysis buffer can significantly reduce the lysate viscosity especially when minimum lysis buffer is required. --Chun
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji Edayathumangalam Sent: Thursday, October 25, 2012 5:55 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resuspension of bacterial cell pellets Hi Folks, Thanks for your responses. To clarify, I have looked into any fluctuations in cell pellet volumes (autoinduction, cell lysis, toxicity) and this isn't such a case. My colleague's cell pellet weights are the standard 3g or so/L and that's why I strongly suspect the resuspension volumes to be an issue. Thanks. Raji On Thu, Oct 25, 2012 at 8:15 AM, Raji Edayathumangalam <r...@brandeis.edu> wrote: Hello Everyone, Sorry for this rather naive and non-CCP4 question but I am very curious. My rule of thumb is to resuspend bacterial cell pellets in about 1-2% of the original culture volume for a wet weight of about 3g of bacterial pellet per L of culture volume. For example, Typically, the total volume of my resuspension for a 6L-bacterial cell pellet is around 60-70mL or about 40mL, if I really try to minimize the volume of buffer. Every protocol I have read over the years seems to indicate something similar. In troubleshooting one of my colleague's protein preps, I found that she is resuspending 6L of cell pellet with a total of pellet+buffer volume of 5mL. In practice, I would not physically be able to resuspend a 6L pellet in 5mL (3g pellet/L culture) without making a very viscous and lumpy soup. My suspicion is that such small volumes are a source of some of her issues, including a high number of impurities in her elution from affinity columns. I'm curious to hear what other folks do and recommend. Cheers, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
