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Extremely small bacterial colonies after transformation


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#1 Agnes85

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Posted 28 April 2010 - 02:18 AM

Hi,
I did ligation and then transformation of E. coli.
Bacteria were grown o/n at 30C (because the construct encodes an eucaryotic protein which is toxic-> this is not my insert).
The plates were: TB (terrific broth) with chloramphenicol. After about 16 h I received only extremely small bacterial colonies (about 30) so I decided to prolong incubation at 30C.

I did miniprep, however I heard an opinion that I shouldn't use this plasmids for further steps (even if they have expected insert) because the bacterial colonies were growing slowly so probably the isolated plasmids slow down the grow rate of bacteria (it wouldn't be good for our future plans). When I repeated transformation with the same ligation I had the same results.
Among the grown colonies I found the right ones (with expected insert).

Has anybody had similar experience (exremely small bacterial colonies after transformation with right plasmid)?
Could anybody help?

#2 RAinsmallpharma

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Posted 28 April 2010 - 11:05 AM

Usually this is because of small colonies of contamination, not transformation.

#3 HomeBrew

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Posted 28 April 2010 - 01:33 PM

...so probably the isolated plasmids slow down the grow rate of bacteria...


Not sure you can conclude that yet...

How big are the colonies of your untransformed recipient E. coli strain after growing at 30C on the same media without chloramphenicol for a similar amount of time?

If you pick a single colony off the original transformation plate, grow it up in broth, dilute the culture, and plate for single colonies, are the resulting colonies still small?

If you plate some of the diluted culture on non-selective media, are the resulting colonies still small?

If you pick a single colony off the original plate, grow it up in broth, and do a mini-prep on it, do you recover your plasmid?

#4 Agnes85

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Posted 28 April 2010 - 10:35 PM

...so probably the isolated plasmids slow down the grow rate of bacteria...


Not sure you can conclude that yet...

How big are the colonies of your untransformed recipient E. coli strain after growing at 30C on the same media without chloramphenicol for a similar amount of time?

If you pick a single colony off the original transformation plate, grow it up in broth, dilute the culture, and plate for single colonies, are the resulting colonies still small?

If you plate some of the diluted culture on non-selective media, are the resulting colonies still small?

If you pick a single colony off the original plate, grow it up in broth, and do a mini-prep on it, do you recover your plasmid?



Many thanks. I will do that.

I am able to recover the plasmid and it seems that it is the right plasmid but I will do additional restrictions to be really sure.

#5 HomeBrew

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Posted 29 April 2010 - 01:52 AM

How big are the colonies of your untransformed recipient E. coli strain after growing at 30C on the same media without chloramphenicol for a similar amount of time?


An even better experiment here would be to see what size colonies are produced by plating your recipient E. coli strain transformed with your vector alone after growing at 30C on the same media for a similar amount of time.

#6 Agnes85

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Posted 29 April 2010 - 10:07 PM

Yeah, thanks.

(actually I have already started working in my current lab so in many cases I just rely on the opinion of a person who has experience with this strain).

#7 Placebosz

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Posted 05 May 2010 - 11:45 PM

Sometimes when agar plate is not moist enough, the colony is smaller than usual.

Hope this will help.

#8 DocFlow

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Posted 20 May 2010 - 02:13 AM

those might be star colonies that form when most of the antibiotics in the agar has been used by the transformant colony.

#9 Agnes85

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Posted 20 May 2010 - 11:49 PM

Not exactly 'cos it is possible to isolate plasmid from them.

#10 swanny

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Posted 23 May 2010 - 07:26 PM

those might be star colonies that form when most of the antibiotics in the agar has been used by the transformant colony.

Not likely with chloramphenicol because it isn't secreted into the surronding media, as amp is.

I'd go with homebrew's various culturing suggestions.

Edited by swanny, 23 May 2010 - 07:26 PM.

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#11 Agnes85

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Posted 23 May 2010 - 10:04 PM

Thanks for opinions.
Actually I did sth similar what HomeBrew suggested and it seems to be fine - growing this bacterial strain at 30 C I receive small colonies even without the plasmid with inserted fragment. So it is strange.

Edited by Agnes85, 23 May 2010 - 10:05 PM.





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