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Chemical competent E.coli always from single colony ?


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#1 pET

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Posted 22 March 2011 - 12:58 AM

Dear all
I have to prepare chemical competent E.colis once again. I use the common calcium chorid method.
Now I wonder if it is essential as described in all the protocols to plate them and start growing them from a single colony ? I see the point with a plasmid bearing clone, but what is the benefit here with empty cells ?
It would be quicker and easier to just inoculate the liquid medium with a bit from the glycerol stock or an aliquot from former preparation of chemical competent cells. Can that be disadvantageous anyhow ?
Looking forward to your opinions
pET

#2 leelee

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Posted 22 March 2011 - 07:15 PM

It means less chance of contamination for one thing. Imagine that your glycerol stock has become contaminated. You then take a loopful allowing this contaminant to grow in your culture together with your desired bacteria. By plating out and selecting a single colony, you avoid carrying over any contaminants.

There may be other reasons that I'm not aware of. But the way I see it, a little extra time now, to save a headache later!

#3 perneseblue

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Posted 03 April 2011 - 10:19 PM

Aside from contamination...which a very very important consideration.

The cells which come out of glycerol stock are unhealthy from the experience and sometimes due to improper freezing, are mostly dead.. Thus direct inoculation of the glycerol stock to culture media can occasionally result in a slow growing culture... as the number of life cells used to start the culture was very small.

Since the time taken to reach the desired OD is longer than expected, you end being unable to do the experiment. Which forces you to re inoculate fresh media and do the experiment the next day..
May your PCR products be long, your protocols short and your boss on holiday




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