ligation and transformation - (Nov/27/2004 )
I think, any antibiotic is not stable for a long period of time at 4°C or even RT... Be sure to use a fresh aliquot. I am careful so that I change aliquots every 2 - 3 weeks in the fridge.
I use kanamycin regularly and keep the stock powder in the fridge (and aliquots of 10mg/ml stock at -20 ). However I recently ordered some from another company and it is kept at room temp.
If your stock is old then maybe it has gone off. Once I have defrosted a stock aliquot I only keep it for 1 week (at 4 degrees) before discarding.
Has anyone else in your lab been using the kanamycin and had a similar effect?
You say that you let the agar cool for 45-60 mins before adding the kanamycin - try cooling it in a water bath at 60 degrees for about 3 hours then add the kan.