Lab 4: Picking a gene for RNAi
In lab today you will obtain a plate containing LB agar supplemented with Ampicillin and Tetracycline cultured with isolated colonies of E. coli containing one of three plasmids of interest on it. Assume that such a colony of thousands of identical cells was formed from a single cell and that cell contains the plasmid which encodes, not only a piece of our genes of interest for RNAi, but also the gene for Ampicillin resistance.
Scan the plate for a single well isolated colony. Once you and your partner have agreed on a candidate colony, check with your instructor to be sure your choice is appropriate. Then you will mark this colony by drawing a circle around the colony on the outside of plastic culture dish on the side containing the media. Be sure to label your plate on the bottom with your initials and lab day. Put it in the refrigerator where you can find it when you come back to make a broth culture.
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 construct to RNAi the gene that you want to study.
- Find your plate in the glass front refrigerator in a rack labeled with your lab day. You and your partner may reassess your choice of the one well-isolated, medium to large colony, selected previously. You may change your selection, if needed. After agreeing upon an appropriate colony, start your overnight culture.
- 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.
- 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 and incubate them at 37°C overnight. Do not forget to make sure the wheel is rotating when you leave!
RNAi Schedule of Experiments
RNAi General Information
Lab 5: Plasmid DNA isolation and transformation
Lab 6: Induction of RNAi plasmid and C. elegans feeding
Lab 7: Single Worm PCR and Agarose Gel Electrophoresis
Lab 8: PCR Reaction Cleanup and Sequencing