Lactose RNAi Plates—Minimal Lactose Media

Protocol by Eric Lambie

Modified by Swathi Arur, Min-Ho Lee and Sudhir Nayak

For 2 liters

1) Make NGM plates as usual

2) Autoclave 30 minutes

3) Let cool to ~47°C in water bath

Add:

• 2 ml 1 M CaCl₂
• 2 ml 1 M MgSO₄
• 2 ml 5 mg/ml cholesterol (in 95% EtOH; bring cholesterol to room temperature on a stir plate and make sure it is completely dissolved).
• 2 ml 100 mg/ml Ampicillin (AMP) (stock concentration 100mg/ml; stored at –20).
• 20 ml of 20% beta-lactose (beta-Lactose can be obtained from Sigma Cat # L8773-250G; lactose is dissolved in sterile water and sterile filtered). Note if beta-lactose is added when the media is too hot, it will breakdown and induction will not work well. Sterile 20% beta-lactose can be stored at 4deg. If it crashes out of solution plate at 37deg for 1hr and it will go back in solution.

4) Pour plates as usual

5) Plates are good for up to 2 months at 4°C

Plating E. coli

1) Grow 2 ml “overnight” by inoculating in LB plus 100 mg/ml AMP in #2059 Falcon tube.

2) Dilute O/N culture 1:100 in LB/AMP and grow 37°C with shaking for 6 hrs.

3) Plate about 150-200ul per NGM/Lactose/AMP plates (these should be warmed at RT before plating the bacteria).

4) Incubate at RT for at least 2-3 days before using.

Conduct experiments!

Enjoy!!