Lab 7: Series 3- Reverse Genetics: Picking Your Transformant
In theory, any colony of bacteria growing on your LB+amp plate should contain a vector plasmid because the gene for antibiotic resistance is not chromosomal, but expressed from your plasmid. Because only transformed bacteria are resistant to ampicillin, if we grow the bacteria on or in a medium containing ampicillin, those bacteria that did not take up plasmid DNA will die while those that express plasmid gene products and transfer the plasmid to their progeny will grow just fine. This process of using a marker (usually antibiotic resistance) to differentiate transformed cells from those not transformed is called selection. Because bacteria reproduce asexually and are immobile on solid media, if you have an isolated colony on a plate, it is likely that the hundreds of thousands of bacteria making up that colony are daughters of a single cell (genetically identical). This allows us to take bacteria from a colony and sub-culture them in liquid media to make millions of identical copies. However, knowing that the bacteria growing in your broth or on your plate with ampicillin all have the vector plasmid responsible for amp resistance, that does not mean that these bacteria also have our gene of interest insert. There are a small proportion of bacteria on your selection plates that have a plasmid without the gene of interest. That can happen when, during ligation, the plasmid DNA annealed back on itself. We need a way to find the colonies that are expressing our gene of interest off the vector plasmid.
To achieve this goal we are going to do a "colony PCR". Instead of adding purified bli-1 or rol-5 gene fragments as template DNA in a pcr reaction with primers specific for your gene (as we did in LAB5), you will add a TINY little part of a colony as the template for your PCR reaction. During the first heat cycle the cells will burst open and release their DNA into the reaction. We will test 8 colonies per group to be sure we continue with bacteria that have the bli-1 or rol-5 gene.
- Obtain a strip of PCR tubes and lids from your instructor. DO NOT separate the tubes!
- Label the side of each tube 1-8
- Find 8 medium to large size, well isolated colonies and circle them and number them 1-8.
- Add 30 ul of master mix to each tube. Your master mix will include: 1X PCR buffer, 10 mM dNTPs, 0.4 uM forward primer, 0.4 uM reverse primer and 2 units/ul Taq.
- After each tube has master mix use a sterile toothpick and gently touch your colony of interest - starting with #1 - and pick up a small amount of the bacteria NOT THE ENTIRE COLONY!!!
- Gently twirl the toothpick in the tube then discard the toothpick
- Repeat until all 8 colonies are picked.
- Snap the lid on the tubes and bring them to your instructor.
| Step || Temperature || Time || Repeat
|| 2 minutes
|| 1 time
|| 30 seconds
|| 30 seconds
|| 1 minutes
|| Steps 2-4 30 times total
|| 10 minutes
|| 1 time
|| end program
Agarose Gel Electrophoresis
After the PCR reaction is completed you will run a gel to analyze the results of the amplification (the search for your gene). You will find a precast 1% agarose gel with Sybr-Green in 1x TAE buffer that is just for your group's use. Make sure that the wells of the gel are closest to the black (negative) electrode and that the gel apparatus has plenty of buffer. Draw a template in your lab notebook so you know which colony is to be put in each lane and which lane contains your DNA ladder. Make sure you get a copy of the DNA ladder key.
Label a set of microfuge tubes with the numbers of your colonies (1-8). Transfer 10μL of each pcr product to the appropriate tube. Add 2 μL of loading dye and transfer all 12μL to a lane of your gel. Place 5 microliters of DNA ladder in another lane. Run the gel for ~ 20min. at ~120 volts. Directions for using the Kodak imager to photograph your gel can be downloaded at: Media: Directions for Using the Kodak imager
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.
- 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.
- 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!
Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.
Links to Labs& Project Info
Lab 11: RT PCR reactions
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Con't
Lab 6: DNA sequence analysis; Mapping Con't
Lab 7: Complete Mapping: Score
Schedule of Reverse Genetics Project
RNAi General Information
Lab 5: Picking your gene to RNAi
Lab 6: Cloning your gene of interest
Lab 7: Picking your transformant
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 10: Scoring your worms and RNA purification