adding antibiotics to already made YT plates - (Oct/24/2006 )
We are having problems with our Kanamycin stocks and I would like to try adding Kan to the top of YT plates. Are there any protocols or suggestions about concentrations or dilutions of the Kan that should be used on the plate. Our Kan stock is at 35mg/ml.
The volume of a typical plate is about 20-25 ml. So, assuming your stock is used at 1 ml/liter, you need 20-25 ul of your stock solution for the agar in the plate. This is too little to spread well, so I would suggest adding 100 ul of sterile water and then spreading the resulting liquid evenly on the plate. Ideally, the plate should sit for a few hours while the antibiotic diffuses and the liquid dries. This works best with slightly dry plates.
phage434
i was just wondering what your stock concentration of Kan is that you use. Also, what concentration do you use when making KAN plates.
We use Kanamycin stock at 35 mg/ml and make up medium with 1 ml of stock solution per liter for a final concentration of 35 ug/ml.
thanks so much for your help.
one more thing, do you do the same thing for amp plates as well?
You can spread ampicillin on plates in the same way. Our stock for ampicillin is at 100 mg/ml, final 100 ug/ml. Incidentally, storing ampicillin in 50% EtOH rather than water allows the stock to be liquid at -20, making it easier to prepare media. This trick does not work for kanamycin, which is insoluble in 50% EtOH.
one more thing, do you do the same thing for amp plates as well?
When you make your plates why dont you simply add the antibiotic to the media before you pour? You can easily make your base media in a 1L bottle (sterilize with a stir bar in the bottle). Once the bottle is cool to touch, add your antibiotic at that time.