BISC 219/F10: RNAi Lab 7

Lab 7: Picking Your Transformant

Colony PCR

To do on the day before the next lab: You and your partner will return to the lab to make an overnight broth culture of your selected colony as described below. This process will create a sub-culture of many identical copies of the plasmid carrying the pL4440 plasmid construct to RNAi the gene that you want to study.

1. Find your plate in the glass front refrigerator in a rack labeled with your lab day. Make sure you can see the colony you selected last lab period.
   
2. Begin by pouring (DO NOT PUT A PIPET INTO THE STOCK LB!!) 10 ml of sterile LB + tetracycline (12.5 μg/ml) broth from one of the stock containers in the refrigerator into a sterile orange-capped 15ml conical tube. You will use the volumetric marks on the tube for measuring the media rather than using a pipet. Make sure the LB stock does not look cloudy (indicating contamination by a previous user) and take care not to contaminate it yourself.
   
3. Add 10 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator). Calculate the effective concentration of ampicillin that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook.
   
4. Replace the cap of your LB +amp broth and invert the tube several times to mix the contents.
   
5. Label two sterile glass culture tubes (found in a rack in the lab) with tape in your team color. Label one with "pL4440 and the gene name" and your initials. Label the other with your initials only.
   
6. Using a 5 or 10 ml sterile disposable pipet, pipet 4 ml of your working solution of LB+ampicillin broth into each of the 2 tubes. Be careful not to touch the tip to anything non-sterile.
   
7. Inoculate the broth with your bacteria by using a sterile toothpick to scrape your candidate colony off the plate. Be sure not to touch the plate with the toothpick except on the desired colony and don’t pick up any satellite colonies. Make sure the toothpick falls into the sterile broth. The second tube of broth labeled with just your initials is a control and should not be inoculated with bacteria as it is your control for contamination.
   
8. Balance the 2 tubes across from each other on the rotating wheel in the incubator at the front of the room when you come in the door and incubate them at 37°C overnight. Do not forget to make sure the wheel is rotating when you leave!
   

RNAi General Information
Media Recipes
Lab 5: Picking your gene to RNAi
Lab 6: Cloning your gene of interest
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 10: Scoring your worms and RNA purification
Lab 11: RT PCR reactions