LB Agar Plates¶
Contributed by Paul Barber
LB agar plates for cloning
Batch makes about 40 plates.
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)
Stock solutions 50 mg dissolved in a small amouth of DMSO 200mg in 10mL dH2O (store at 4 in 1mL aliquots) use 100mM 238 mg IPTG in 10mL dH2O (store at –20 Ampicillin 20mg/mL 50uL on each plate IPTG (VWR EM-5800) 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
- Add 250 mL of dH2O to a graduated cyclindar.
- 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
- Mix powder well to bring into solution
- Add dH2O to total volume of 500 mL and transfer to 1 L flask
- Put on stirring hot plate and heat to boil for 1 min while stirring.
- Transfer to 1 L pyrex jar and label with autoclave tape.
- Autoclave at liquid setting for 20 minutes in a basin making sure to loosen top
- Let agar cool to ~55C (you should be able to pick up the jar without a glove)
- Add your antibiotic at this point.
- Make sure bench top has wiped down with bleach/EtOH.
- Remove sterile Petri dishes (VWR 25384-208) from plastic bag.
Save the bag for storage
- 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.
- Swirl plate in a circular motion to distribute agar on bottom completely.
- Let each plate cool until its solid (~20 minutes) then flip so as to avoid
condensation on the agar.
- Store plates in plastic bags in fridge with: name, date and contents
Note any additive
This method is based, with permission, on an original protocol available here.