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colonies grow in amp plates not in broth - (Feb/03/2008 )

hi all,
I am facing a strange problem, my colonies (ligation) are growing in plates but not in broth. Pure plasmid transformed colonies is growing in both plate and broth. I read up all the archives in the topic, but cannot really understand what has happened. I tried with two different ligation mix, one had pCDNA3.1 and another pBSK+. I have already tried three times. the resistance marker is ampicillin. I have already changed amp plates, earlier had been using plates made two weeks before with amp stock stored in -20 in 10mg/ml stock. I used 100ug/ml in plate.Now I have made new plates from new freshly ordered stock. now in these new plates no. of colonies have reduced but again they are growing very slow in culture in the same conc i.e. 100ug/ml.
I doing miniprep today, let's see if I get the right plasmid.
what do you think is happening.



is there anything special with your insert (toxic, unstable, high repetitive...)?


your old amp was degraded. the fresh stock is giving you more amp in your plate (and culture) than was the old stock.


no the protein is not toxic, it EGFP.
and yes perphaps that was the case as mdfenko suggested the amp might have been degraded.
today I checked whether the ligation was happening in gel and yes it is working.
so what is happening


How are you innoculating the broth?

Are you giving it long enough to grow?


QUOTE (MTO @ Feb 5 2008, 11:39 PM)
How are you innoculating the broth?

Are you giving it long enough to grow?

It is overnight culture. You know something very interesting is happening with one of my clones.
I somehow managed to get a positive clone. The colony was a huge one on a freshly prepared plate from fresh amp stock but when I kept in liquid culture O/N. the growth was half of what I actually get. The backbone of this clone pBSK(+) and insert is EGFP. And you know what my other constructs which were prepared earlier are growing fine. No problem at all. But this one is showing same pattern again and again. What happened? Did the copy no. get reduced? Is this possible?

can anyone tell me what is happening? blush.gif


if the EGFP is being expressed in the e coli, then the protein could become toxic by virtue of filling up the cell and getting in the way.

IF this is a toxicity problem, try growing your cells at a lower temperature about 30 celsius.

Copy number can be reduced...
if the origins of replication is damaged (very unlikely)
if you are meeting one of those strange DNA sequences which e coli does not like. (Probably not the case here)