Kanamycin - quality of kanamycin (Jan/12/2007 )

Hi all,


It has been several times that the quality of the kanamycin that I used for plasmid selection shows discrepency between providers where I bought it and also problems due to some time storage problem which has a real influences on its quality: so that, when I think that I had 25 µg/ml, my real concentration is less than that and sometimes contaminations arise!

So I would like to get some feedback from others lab/persons who are routinely using kanamycin for selection and if they have noticed these kind of problems and what they are doing to fix them.

Also do you think that using stronger concentrations of kanamycin such as>=50µg/ml would be valuable to reduce the loss of stability/quality of the kanamycin? And maybe using such high concentrations would require a more active processing enzyme KAN on the vector to hydrolyze these strong kanamycin concentrations?

thanks for your help and comments


Djin

Hi Djin,
I work sometimes with kanamycin. I've heard that kanamyin-sulphate is the best you can use. You get this in powder and you can make a solution. The rest of the powder form keeps its activity for a long time.
You should avoid freezing and thawing so it's best to aliquot it in a couple of eppendorf cups. Also avoid that it becomes warm.
I think that raising the concentration can be a bit tricky, you don't exactly know the activity and there is a chance that you will use too much. And when something is losing its activity, it keeps on loosing it. So you are not able to check for a good concentration now and keep on using thing concentration.

Hi Djin,
we use kanamycin-sulphate 25 µg/ml and never had any problem with contaminations. If I had doubts about quality of my antibiotics I would probably buy a new one and kept storing it properly. Kanamycin is quite cheap.
Anyway if you don´t want to buy new one, you could raise the concentration to 50 µg/ml and see if bacteria with the selection marker grow on it.

We also had problems with Kanamycin in the past when we bought it from a cheaper supplier. Since we moved back to Sigma we haven't had any problems, but we also now aliquot it out instead of continually freeze/thawing. We also use it at 50 ug/ml without any problems.

How do you make up your Kan?
Our lab uses a 50mg/ml stock in water and filter sterilize it to store in the 4C. We use 50ug/ml for solid media and 25ug/ml for liquid. We had some problems last year, but it was lack of bacterial growth as opposed to contamination. I think it was a problem with the bacteria/transformations rather than the Kan though.

we normally keep our antibiotics in -20C. alliqout it into 1.5ml tubes.
it works well for quite a long time.