2 replies to this topic

[spn25]

Our lab has recently suffered from protein expression problem..

We manage to get the overnite cultures(5ml of 2xYT plus a single colony plus 5 ul of ampicilin) inoculated and the products seemed to be coludy enough.
However, when we try to inoculate the overnite into 1 L of media, the media first turn cloudy, OD reaches 0.2 or less, and then decreases, with visible clumps of cell debris precipitated. As a result, we could not carry out the further induction at all.
The most annoying part is that this happens on and off, for example, prearing 10 flasks, 8 were affrected by the problem and 2 were OK...
This renders great pain and puzzle in trobleshooting the issue
We tried to clean the bug room with virkon and the problem still exists.....
Could anyone give us some advice?

Edited by spn25, 02 October 2004 - 03:46 AM.

[leahf]

Hi
sorry but i have no solution here...just a thought:

it may be useful to know if your problem really is virus contamination.

you can do a standard viral titration for your affected flasks - prepare soft agar, use one of the cultures that did grow right as an indicator strain (that is, add it to the soft agar at a high enough concentration that it would form a lawn after poured into plate) and then add, at varying concentrations, samples from your affected flasks. viral contamination should show up as plaques in the lawn formed by the indicator strain.

then you know if it really is viral, to concentrate on decontamination and if not then  to look elsewhere...

[carles]

perhaps wash your flasks and pipettes with a solution of 2% hypochlorite for 1 hour  following extensive washing and then autoclaving