Lab 8: Series 3- Creating the feeding strain of bacteria for RNAi in C. elegans
To do on the night before this lab:
You and your partner will return to the lab to make an overnight broth culture of one of the colonies that you are sure contains the gene of interest (determined from your visualization of successfully amplified, appropriately sized DNA seen on your gel photo). The sub-culture you will set up tonight will create many identical copies of bacteria that carry the plasmid containing your gene of interest.
- Find your LB+amp plate in the glass front refrigerator in a rack labeled with your lab day. Make sure there is some bacteria remaining on the plate of a colony that you saw successful gene amplification in the pcr product.
- Begin by pouring (DO NOT PUT A PIPET INTO THE STOCK LB!!) 10 ml of sterile LB 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.
- 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.
- Replace the cap of your LB +amp broth and invert the tube several times to mix the contents.
- 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.
- 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.
- 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.)
- 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.
- Incubate these broth cultures at 37°C overnight. Do not forget to make sure the wheel is rotating when you leave!