Protocol Code: M2

LB (Lysogeny broth)

(Taken from Wessel Lab at Brown University)

The following protocol makes ~40 plates.

Making the broth

1. Add 250 mL of dH₂O to a graduated cyclindar.
2. Mix one of the following:

1. Mix powder well to bring into solution
2. Add dH₂O to total volume of 500 mL and transfer to 1 L flask
3. Put on stirring hot plate and heat to boil for 1 min while stirring.
4. Transfer to 1 L pyrex jar and label with autoclave tape.
5. Autoclave at liquid setting for 20 minutes in a basin making sure to loosen top
6. Let liquid cool to ~55C (you should be able to pick up the jar without a glove)

Pouring 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 storage).
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 additive).

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