A few weeks ago I described some problems we were having with cosmid
libraries. I got several good responses. The one below was the best and
might be useful to others.
"What you have been seeing is actually the gradual loss of the
selection pressure, which results from the degradation of the
ampicillin in the medium by the beta-lactamase produced by the amp-
containing clone. This eventually leads to the loss of the amp-
containing cosmid. You might recall the growth of satellite colonies
around the genuine transformants when you let the incubation of
transformation plates go longer, say two days instead of overnight.
After 4-6 weeks the clone has lost the amp-containing cosmid and
becomes ampicillin sensitive. That is why you transferred the cells to
ampicillin-containing liquid medium and you did not see any growth
the next day. The same goes for the lower yield of cosmid DNA after
3-4 weeks, since the colony contains a population of resistant and
sensitive cells. My suggestion to you is to make a frozen cell stock.
This can be easily achieved by saving 1-2 mL liquid cell culture when
you first prepare the cosmid DNA from freshly streaked plate. It is NO
NEED to pick up a single colony for your cosmid preparation each
time. After you have checked and know that the cosmid DNA is good,
spin down the cells, discard the supernatant and resuspend the pellet
in 1 mL sterile 20% glycerol solution (better to prepare two
microtubes of the cell stock). Store the cell stock at -80 C. The cells
are viable for years. Whenever you need to propagate cells for cosmid
preparation, just get a loopful or 5 uL as an inoculum.
Southern Regional Research Center
1100 Robert E. Lee Boulevard
New Orleans, LA 70124
On 24 May 1995, Barry Bowman wrote:
> We have been working with the Sachs/Orbach cosmid library for Neurospora.
> In the last year we have had problems with maintaining viable colonies. We
> streak-out a clone from the library on LB/amp plates and then use these the
> next day to make a "stock plate". The "stock plate initially has viable
> cells and we can make good cosmid DNA from them. However, after 3-4 weeks
> the yield of cosmid DNA from cells derived from the stock plate drops
> dramatically. After 4-6 weeks the we can't even grow the cells after
> transfer to liquid media.
>> Has anyone else had this problem? Are we leaving something out of the
> media? Are the cosmid-containing cells less viable?
>> Barry Bowman - U. of California, Santa Cruz