Dh5 alpha cultures not viable ?
stacyef at stacyef.net
Fri Feb 24 23:04:24 EST 2006
In article <mailman.1224.1140796133.29584.methods at net.bio.net>,
"Siddhartha Parida" <sidharth4 at gmail.com> wrote:
> I had ordered a culture of DH5alpha from a supplier and when i
> inoculated it in a LA + Amp (10mg/ml) the culture was able to grow, i
> streaked it into slants and keep it at 4C for a month but now when i
> want to work on it again, the culture is not growing , i have repeated
> ot many times but without sucess.
> Point to be noted: That our water bath shaker is out of order so i had
> kept the flask containing the media in a bacterialogical incubator at
> 37C, is vigorous shakes a must for revival of these culture ?
Unless you transformed your bugs with an AmpR plasmid, they should have
died in ampicillin-containing medium in the first place. If you bought
DH5a, didn't transform it and and it grew in Amp broth then something is
wrong. Either you were sent the wrong strain or there was something
wrong with your broth. If you added the Amp to the broth weeks or months
before using it, the ampicillin may have deteriorated. It's not suitable
for long-term storage in that form. You either need to add it to your
broth fresh and replace it with a fresh batch every couple of weeks or
you need to use a more stable drug in its place (carbenicillin is more
stable and the shelf life of pre-poured petri dishes and broths is
longer). Once you streaked it onto a slant, it may have died at that
point because the ampicillin was still active in that medium.
I would never try to salvage an Amp-sensitive strain from a dead culture
mistakenly plated on Amp media. Anything you recover may be a
contaminant or worse, a DH5a cell that picked up a functional beta
lactamase gene one way or another. You could be unintentionally
selecting for a mutant. Since DH5a is a commonly used host for AmpR
vectors, having a mutant that's already resistant to ampicillin would be
a nightmare for most people who use it.
If you are trying to recover an AmpR plasmid that WAS supposed to be in
that DH5a (which you should rename so no one in the future makes the
mistake of thinking they're getting an Amp-sensitive strain), you can
recover plasmids from dead cultures. The yield is extremely low (often
undetectable on a gel) but if you use it for transformation, you don't
need to recover much.
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