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Weird results with bacterial transformation

Contamination

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5 replies to this topic

#1 rmbio

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Posted 16 May 2015 - 12:00 AM

Hi all

 

I have been doing cloning and transformation almost since past 10 years, but have never experienced the following. In the lab which I have newly joined, my student is trying make the competent cells. While checking the efficiency by transforming pUC18, she is getting some background contamination. This sometimes looks like a mat growth made up of millions of tiny colonies onto which we get larger E. coli colonies. Few other times we get two type of colonies-larger and smaller; I presume that the larger ones are transformed E. coli. When only water is transformed containing no plasmid, no growth is seen suggesting that the contamination is not being introduced during the transformation experiments and that ampicillin which is being used is fine. Even after switching to new plasmid which was newly procured and untouched, same results were observed. Since negative control (water transformation) is fine, I am really wondering what might be going on wrong!!

 

Has anyone experienced the same thing any time?

 

Thanks

 

NB: I have also tried making the competent cells again (from another strain brought from a different lab) which has given the same results.


Edited by rmbio, 16 May 2015 - 12:10 AM.


#2 perneseblue

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Posted 16 May 2015 - 05:50 AM

Was the water transformation control done exactly the same as a plasmid transformation? Did you do both experiments side by side?

 

Perhaps the SOC was contaminated?

 

I know of one bench in my lab that if you do cell plating work there, you will get alot of yeast contamination. It is probably due to the presence of an air vent just above it.


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#3 r.rosati

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Posted 16 May 2015 - 06:59 AM

My best bet is that the antibiotic isn't working (it's below the optimal concentration), so it stunts, but doesn't block, the growth of untransformed bacteria.

 

If the effects range, then perhaps the stock antibiotic is ok, but your student is adding it while the agar is very hot, and the antibiotic degrades (at varying degrees depending on how hot the agar was at the time). You're using pUC18 so adding ampicillin, and the beta-lactams are very sensitive to heat inactivation.

The recommended temperature for adding ampicillin to the medium is 55°C or below. That's roughly when you can keep your hands on the bottle for more than 30 secs without feeling the urge to take them off (of course you know that by experience you just get the feeling). Just in case, tell your student that being able to hold it for 3 seconds doesn't count wink.png


Edited by r.rosati, 16 May 2015 - 07:15 AM.


#4 rmbio

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Posted 19 May 2015 - 12:14 AM

Thank you perneseblue and r.rosati for your replies.

perneseblue, yes, both water and plasmid transformations were done EXACTLY in the same manner at the same time.  

r.rosati, the thing is that I am not getting any growth with water transformation. If ampicillin would have gone bad or the concentration is wrong, I should have got growth in water transformation also, isn't it?



#5 r.rosati

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Posted 19 May 2015 - 03:13 AM

I see. Well, what you describe is really, really similar to what you'd expect from an insufficient amount of antibiotic, though. And when ampicillin is low, given enough transformed colonies on a plate, they'll produce enough beta-lactamase to reduce antibiotic pressure even further and possibly create a lawn of untransformed colonies. While when the transformed colonies are few in number, then what you see are satellite colonies around them, and no colonies away from them. Does this match what you see? If not, then I concur it must be something else.

But would you humor me and double-check the ampicillin stock (calculations, initial purity etc) and the temperature at which it's added to the medium?


Edited by r.rosati, 19 May 2015 - 05:00 AM.


#6 phage434

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Posted 19 May 2015 - 05:23 AM

Another thing to check is how fast these small colonies arise.  As r.rosati suggests, beta lactamase will be excreted by resistant colonies. Ampicillin is bacteriostatic and will not kill bacteria, so growth may be possible of satellite colonies.  Usually this happens after more than a day of growth, not overnight.







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