Hi Jacob, take some (few ul) of the lysed culture and spread that together with regular cells onto an agar plate so that you (would) get a lawn of bacteria after O/N incubation. If it is phage you'll get clear plaques in your lawn, very distinctive. If it is regular cell lysis induced by the toxicity of your protein you won't get plaques. We have dealt with phage contamination and the best and most reliable way to get rid of them is not to grow any cells for a while (at least a week). This is a time to do a lot of cloning..... Also, we now have installed large UV lamps in the room where we grow our cells so that we can irradiate that room regularly.
Good luck, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -----Original Message----- From: CCP4 bulletin board on behalf of Jacob Keller Sent: Thu 7/2/2009 2:05 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Shredded E coli pellets Okay, it seems that the consensus is phage infection. Is there anything to seal the diagnosis? Also, does anybody have literature on de-phaging glassware? I am assuming that regular autoclaving will not do the trick? Jacob ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ******************************************* ----- Original Message ----- From: "Jacob Keller" <j-kell...@md.northwestern.edu> To: <CCP4BB@JISCMAIL.AC.UK> Sent: Thursday, July 02, 2009 11:42 AM Subject: [ccp4bb] Shredded E coli pellets > Dear crystallographers, > > I recently expressed some new constructs, and found after my usual > expression protocol that the cell pellets were not compacted at the bottom > corner of the bottles us usual, but were instead smeared as a film on the > side, and further, were somewhat clumpy, like clots, and with a smaller > pellet in the usual location. The centrifugation was exactly as usual. I > noticed that there was also a bit more foam in the medium than usual, but > I am not convinced that this was the issue, although it might be a > symptom. My suspicion is that the constructs are lethal and cause cell > lysis, but I am not sure. Has anybody seen this phenomenon before, and > gotten to the bottom of it? > > Jacob Keller > > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > Dallos Laboratory > F. Searle 1-240 > 2240 Campus Drive > Evanston IL 60208 > lab: 847.491.2438 > cel: 773.608.9185 > email: j-kell...@northwestern.edu > ******************************************* >
