RNA Interference (RNAi) Plates
Adapted from: Eric Lambie, Dartmouth College, Hanover, NH
Reagents (for 1L):
- NaCl (Fisher, BP358-212) - 3 g.
- Peptone (Fisher, BP1420-500)- 2.5 g.
- Agar (Sigma, A7002-500G) - 17 g.
- DI Water - 975 ml
- 10mg/ml Cholesterol (Sigma, C75209-100G) - 0.5 ml
- 2mg/ml Uracil (Sigma, U0750-25G) - 1 ml
- 1 M CaCl2 - 1 ml
- 1 M MgSO4 - 1 ml
- KP Buffer (ph 6) - 25 ml
- 100 mg/ml Ampicillin – 1 ml
- 20 % β-lactose – 10 ml
- Start by adding about half of the DI water to an Erlenmeyer flask.
- Stir the water while adding the NaCl and Peptone first. Add both slowly to avoid clumping.
- Once both are dissolved completely, add the remaining volume of water and the agar to the solution. Keep the solution mixing the entire time.
- Cover the top of the flask with foil and autoclave the solution for 30-60 minutes using a liquid cycle. Do not take the stir bar out; it will be used later while pouring plates.
- Once autoclaving is complete, cool the solution to 56-58 °C with constant stirring. There are 2 ways of doing this:
- Place the flask in a 56°C water bath and let sit for an hour, or
- Perform a "Quick Water Immersion Cooling (QWIC)." NOTE: This technique works well at quickly cooling the solution uniformly and is utilized in our lab. It is not "scientifically proper," but it works well. Consult your professor first to see if this technique is appropriate for your lab.
- Keep the top covered with foil in order to maintain sterility. (Figure 1).
- Add the remaining reagents to the solution. Make sure to practice sterile technique with each addition. Open the top and add the reagent; do not completely remove the foil. (Figure 2) Immediately recap the top with foil once each addition is complete. (Figure 3)
- 0.5 ml cholesterol
- 1 ml uracil
- 1 ml CaCl2
- 1 ml MgSo4
- 25 ml KP buffer
- 1 ml 100 mg/ml Ampicillin
- 10 ml 20 % β-lactose
- Pour 10 ml of the solution into 60x15mm plates. The plates should sit overnight, allowing them to harden. 1 liter of solution should pour between 90-100 plates.
- Once dry, the RNAi plates should be stored at 4 °C.
Keep the flask covered after removing from the autoclave.
Remove cap only when adding reagents.
Re-cover with foil after each reagent has been added.