LB Agar Plates

Published on Author Giorgio Gilestro

Batch makes about 40 plates.
Making the LB Agar

  1. Add 250 mL of dH2O to a graduated cylinder.
  2. Weigh out 20g of premix LB Agar powder (VWR DF0445-17) or:
    5.0 g tryptone
    2.5 g yeast extract
    5.0 g NaCl
    7.5 g agar
  3. Mix powder well to bring into solution
  4. Add dH2O to total volume of 500 mL and transfer to 1 L flask
  5. Put on stirring hot plate and heat to boil for 1 min while stirring.
  6. Transfer to 1 L pyrex jar and label with autoclave tape.
  7. Autoclave at liquid setting for 20 minutes in a basin making sure to loosen top
  8. Let agar cool to ~55C (you should be able to pick up the jar without a glove)

Pouring the Plates

  1. Make sure bench top has wiped down with bleach/EtOH.
  2. Remove sterile Petri dishes (VWR 25384-208) from plastic bag (save the bag for
  3. Pour a thin layer (5mm) of LB Agar (~10mL) into each plate being careful to not lift the cover off excessively (you should be able to just open up enough to pour).
  4. Swirl plate in a circular motion to distribute agar on bottom completely.
  5. Let each plate cool until its solid (~20 minutes) then flip so as to avoid condensation on the agar.
  6. Store plates in plastic bags in fridge with: name, date and contents (note any

Special Additives (to be added to LB Agar right before pouring plates)

Ampicillin (VWR 80055-786)
50 mg dissolved in a small amout of dH2O
(concentration 100 ug/mL)

X-gal (VWR IB02260)
50 mg dissolved in a small amouth of DMSO

Stock solutions

Ampicillin 20mg/mL
200mg in 10mL dH2O (store at 4 in 1mL aliquots) use 50uL on each plate
IPTG (VWR EM-5800) 100mM 238 mg IPTG in 10mL dH2O (store at –20 in 1mL aliquots) use 40uL on each plate
X-gal 40 mg/mL 400 mg X-gal in 10mL DMSO (store at –20 in 1mL aliquots foil wrapped tubes) use 40uL on each plate