1. K-State home
  2. »Division of Biology
  3. »Herman Lab
  4. »Protocols
  5. »Antibiotic Usage

Herman Lab

Antibiotic Usage

  • How to make LB liquid plus antibiotics:
    • Ampicillin – The frozen stock solutions of ampicillin are at 50mg/ml and 100mg/ml in H2O, and are marked with a red sticker.   The final concentration for LB liquid culture is 50ul/ml.  To obtain this in 100ml (the amount in each LB bottle), add 100ul stock solution.  
    • Kanamycin – The frozen stock solutions of kanamycin are at 50mg/ml in H2O, and are marked with green.  The final concentration for LB liquid culture for growing plasmids is 50ug/ml, and for cosmids is 20ug/ml.  To obtain 50ng/ml in 100ml of LB, add 100ul stock solution, and to obtain 20ug/ml, add 40ul stock solution.  
    • Tetracycline – The frozen stock solutions of tetracycline are at 15mg/ml in methanol and are marked with black.  The final concentration for LB liquid culture is 15ug/ml.  To obtain this in 100ml of LB, add 100ul stock solution.
    • Chloramphenicol – The frozen stock solutions of chloramphenicol are at 25mg/ml in 100% ethanol and are marked with purple.  The final concentration for LB liquid culture is 25mg/ml.  To obtain this in 100ml of LB, add 100ul stock solution.
  • How to make LB plates plus antibiotics:
    • Follow the recipe card in box for making LB plates, being sure to add the agar.  After autoclaving, and when the agar has cooled enough that it's not too hot to touch (about 1 to 1.5hrs), add antibiotics as follows:
      • Ampicillin – add 1ml ampicillin (at 100mg/ml) per liter of agar to obtain a final concentration of 100ug/ml.  Mark the plate with a single red line on the side.
      • Kanamycin – add 1ml kanamycin stock (at 50mg/ml) per liter of agar to obtain a final concentration of 50ug/ml.  Mark the plates with a single green line on the side.
      • Tetracycline – add 1ml tetracycline stock (at 15mg/ml) per liter of agar to obtain a final concentration of 15ug/ml.  Mark the plates with a single black line on the side.
      • Chloramphenicol – add 1ml chloramphenicol stock (at 25mg/ml) per liter of agar to obtain a final concentration of 100ug/ml.  Mark the plates with a single purple line on the side.  
  • How to determine volumes to use to obtain a certain concentration:
    • Use the following formula to determine amount of stock solution to use for any amount.  Make sure units match!
      • C1V1 = C2V2         
      • C = concentration; V = volume
      • Example:  You have a stock solution of A at 100mg/ml.  You want 150 ml at 100ug/ml.  Use the C1V1 for what you want, and the C2V2 for what you have.  Just plug in the numbers:
      • (100ug/ml)(150ml) = (100000ug/ml)(x)
      • 15000 = 100000x
      • x = 0.15ml, or 150ul
      • Remember that you have to convert mg/ml to ug/ml, which you can do by multiplying by 1000.