Kuperwasser Lab | Making Selective Plates

Materials

– LB powder (BD #241420)

– Agar powder (BD #214010)

– sterile Petri dishes (VWR #25384-302)

– ddH₂O

– autoclaveable glass bottle

– scale, weigh boats, etc

– stir plate and stir bar

– autoclave, autoclave tape, gloves

– water bath set to 50^oC

– drug for selection

– tips, tubes, etc

Procedure

Determine how much LB-Agar you wish to make (one plate ~ 25mL)
Select glass bottle with a volume at least 2X the amount of LB-Agar you wish to make
Add LB powder to bottle (15.5 g/L)
Add needed volume of ddH₂O to bottle and mix on stir plate (should mix thoroughly)
Add Agar powder to bottle (15 g/L) and mix on stir plate (should appear cloudy and will not fully mix until autoclaved)
Autoclave on “LIQ” cycle (takes about one hour)
Move bottle to water bath until it is cool enough to hold without gloves
1. While waiting for bottle to cool, label plates and thaw antibiotic
2. Calculate amount of antibiotic you will need for each bottle. Antibiotics are usually at 500X (e.g. use 500μL kanamycin for 250mL LB-Agar)
Vortex antibiotic and add to bottle
Pour into plates and leave @ RT with lids halfway on until Agar hardens
If not using plates immediately, store @ 4^oC

Making Selective Plates