Re: [ccp4bb] what happens when freezer goes down
Jackie, We grow cells routinely and freeze pellets after fermentation. In general, proteins are fairly stable until you break cells so you are probably ok unless your protein is very heat labile and it sat at room temperature for hours. However as I mentioned, there is a kind of buffering from the cell that can stabilize the protein until you are ready to use it. The glycerol stocks are probably ok as well since you need just a small inoculum to get your culture growing. The plasmids may be the most affected, but you can sequence if you have aberrant expression. Best, David -- David L. Blum, Ph.D. Director, Bioexpression and Fermentation Facility Department of Biochemistry and Molecular Biology University of Georgia 120 Green Street room A414A Athens, GA 30602 http://bff.uga.edu/ b...@uga.edu On Fri, Apr 25, 2014 at 12:43 PM, Jacqueline Vitali jackie.vit...@gmail.com wrote: Dear colleagues, I know this is not related to ccp4 but I am in need of an answer and many of you work with cells etc. My building had a major malfunction of electricity and the power backup did not kick in. My -80C freezer was without power for over 24 hours until I found out. Because it is small, it goes fast to room temp. I had many glycerol stocks in it with cells, cells with plasmids etc. as well as cell pellets. I am trying to rescue things. My question is what happens to cell pellets. I had many as I like to start purification at the cell pellet level. Are these destroyed when they go to room temp for 24 hours or are they ok? Thanks. Jackie Vitali Cleveland State University
Re: [ccp4bb] what happens when freezer goes down
Hi, My condolences. This is not a fun experience! Your plasmids will be OK :) Glycerol stocks may be rescued as long as after going to room temperature they did NOT get frozen again. Re-freeze will greatly reduce viability - but even after one re-freeze you should be able to streak the glycerol stocks onto appropriate plates and get a few (or more) colonies. Notably this does not mean that expression from these rescued glycerols will be good; furthermore there are quite a few protein expression constructs in our labs that do not store well as glycerol stocks - fresh DNA transformation is the only way to get consistent expression for them. Proteins in frozen cell pellets: that's iffy. The outcome greatly depends on the protein itself - for instance I had several proteins that had activity loss issues even when stored in LN2. In a -80 freezer they would lose 40-50% activity in a week, and a single freeze/thaw/freeze would kill them instantly (even in cell pellets). Then again, other proteins are fine 'no matter what'. In cases like yours it really helps to have some sort of parameter you can follow (activity, or something). Frozen protein samples - can be even more iffy than pellets. Pellets are generally cheap to make, though. Unless they are isotope-labeled or suchlike. Artem - Cosmic Cats approve of this message On Fri, Apr 25, 2014 at 11:43 AM, Jacqueline Vitali jackie.vit...@gmail.com wrote: Dear colleagues, I know this is not related to ccp4 but I am in need of an answer and many of you work with cells etc. My building had a major malfunction of electricity and the power backup did not kick in. My -80C freezer was without power for over 24 hours until I found out. Because it is small, it goes fast to room temp. I had many glycerol stocks in it with cells, cells with plasmids etc. as well as cell pellets. I am trying to rescue things. My question is what happens to cell pellets. I had many as I like to start purification at the cell pellet level. Are these destroyed when they go to room temp for 24 hours or are they ok? Thanks. Jackie Vitali Cleveland State University
