I gave overnight culture from my DH5alpha glycerol stock in 5 ml LB and 5 ml LB+Kanamycin as control.There was no growth in the antibiotic added LB the next day(validating that my stock isn't contaminated with a antibiotic resistant plasmid).The next day I gave 1% subculture from the 5 ml LB culture to 100 ml media and prepared competent cells using the traditional CaCl2 MgCl2 method and also performed transformation with a kanamycin resistant plasmid to test their efficiency.The problematic thing is that I got four positive colonies in my control plate(Kanamycin agar plate where just the DH5alpha competent cells without any plasmid) was plated.It is very difficult for me to trace the contamination because I did each of the step in laminar hood and also near flame and I had autoclaved everything from tips,glycerol,media,CaCl2,MgCl2 before using.Can anyone tell what might be the possible source of contamination in this case?
Could it be some issue with the plates rather than with the bacteria? If no contamination in the cells is seen in liquid LB medium + Kanamycin, are you sure that the Kan is still active in the plate? Maybe it could be an old plate, or maybe you added the antibiotics when the medium was still too hot?
If that's the case, it's just some DH5 still able to grow...
My guess is those 4 colonies are probably not E. coli and just some environmental contaminant that was introduced. Pipets and pipet tips are a common source of introducing low levels of contamination (most people don't clean their pipetors) especially if not using barrier tips.
If you used a bottle of LB that had been used previously it may have had prior KanR strain bacteria present. Check to see if the colonies look like E. coli, S. aureus is another common contaminant (similar colony size but slightly different color than E. coli)
Amy Klocko I used freshly prepared autoclaved LB that was not opened before and even freshly prepared plate.The most confusing thing to me is that in liquid LB there is no growth on addition of kanamycin but in kanamycin agar plates the same competent cell is showing growth
Even in a laminar flow hood, contamination can happen. Bacteria from cross-contaminated micropipettes, your sleeves, etc. Especially since you are in a lab that uses bacteria and plasmids. Just takes one person "borrowing" the sterile pipet tips and you can have a problem.
It's also possible that the colonies are NOT E. coli and are another bacteria.
Another possibility is mutation. Kanamycin isn't a particularly strong selection. A single point mutation in the 16S rRNA gene can give resistance.
This sort of "background noise" is really common. You'd sequence any potential transformed colonies anyway.
I'd say just continue with your experiment.
Katie A S Burnette another thing that is worrying me is contamination of my DH5alpha glycerol stock.On two simultaneous days,I am getting different results from the same stock.On day 1 there was no growth in kanamycin,on day 2 there was growth in kanamycin.
Very unlikely, but DH5alpha cells are common and cheap. Buy new ones and don't waste your time trying to solve something this minimal. You have either contamination from the environment, mutation, or expiring antibiotics.
You've already spent more time than necessary worrying about 3 unexpected colonies. It happens, just continue your project.
@Namrata
Please, may I know whether this problem was later solved and what was the solution? Bcos I currently have the same problem with my cloning. Thanks
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