Lab 8: Series 3- Creating the feeding strain of bacteria for RNAi in C. elegans
In order for RNAi to be successful we must insert the plasmid expressing dsRNA of our C. elegans gene of interest into the bacteria that will make lots of double stranded RNA. We will then feed the bacteria that is full of double stranded RNA specific to our gene of interest to our worms. The process of inserting the plasmid into bacteria is called transformation. Transformation, either chemically or electrically, opens up pores or channels in the bacterial membrane that allows for uptake of the plasmid DNA.
Precise pipetting is required for transformation to be effective. To ensure we are all pipetting properly and that our pipettes are properly calibrated follow the procedure below.
Part 1:Calibration of Micropipettes
Before we begin our molecular genetic experiments, we will practice (AGAIN) properly handling and using the micropipettes.
- To calibrate your P1000, P200, and P20 micropipets, label 6 microfuge tubes (1-6) and weigh them. Record the weights in the table below.
- Following the table below, pipet the specified volumes into the pre-weighed microfuge tubes prepared above and then re-weigh them. Record all weights.
- Calculate the weight of the water in grams. 1000 microliters of water should weigh 1 gram at room temperature.
- If the water in any tube weighs more or less than 1 gram, ask your instructor for help. If your calibration is significantly off after several repeated attempts, your pipet (or your technique!) may need adjustment.
| Tube #
|| Tube Pre-Weight
|| Vol. in μL using P20
|| Vol. in μL using P200
|| Vol. in μL using P1000
|| Weight of Tube + Water in grams
|| Weight of Water in grams
|| 200 (5 times)
|| 20 (5 times)
For a Word™ format protocol: Media:Protocol for Micropipet Calibration.doc
Part 2: Measuring the concentration of plasmid DNA using the NanoDropper
Proper transformation requires the concentration of bacteria to plasmid DNA to be right. We will use a nano-dropper to determine the concentration of the plasmid DNA from a previous plasmid isolation procedure.
Use the ThermoScientific NanoDrop Spectrophotometer in L308 to measure DNA by taking Absorbance at A260nm. This spectrophotometer uses only 1 microliter of sample and does not require cuvettes. The sample is held in place by fiber optic technology and surface tension that holds the sample in place between two optical surfaces that define the pathlength vertically and dynamically. Measurement can be assessed in a range of 2 to 3700nm/microliter dsDNA. These are expensive machines so make sure you follow the directions carefully and ask your instructor for guidance as needed.
More information is available from the manufacturer's website at: | http://www.nanodrop.com/HowItWorks.aspx
Using the Nanodropper
1. Clean the upper and lower optical surfaces of the sample retension device by pipetting 2 microliters of clean deionized water onto the lower optical surface. Close the lever arm and tap it a few times to bathe the upper optical surface. Lift the lever arm and wipe off both optical surfaces with a Kimwipe.
2. Open the NanoDrop software from the Desktop of the computer and select the nucleic acids module.
3. Initialize the machine by placing 1 microliter of clean deionized water onto the lower optic surface, lower the lever arm, and select initialize from the NanoDrop software. Once initialization is complete (~10sec.), clean both optical surfaces with a Kimwipe.
4. Perform a blank measurement by loading 1 microliter of purified deionized water.
Note that as in traditional spectroscopy, the blank will be subtracted from subsequent measurements. If you want to determine the contribution of a specific buffer or diluent, measure the buffer first using distilled water as a blank. If the buffer does not contribute to the A 260nm reading, then deionized water is fine to use as the blank. The water or buffer should always be measured to be sure that the instrument has been zeroed properly. The measurement of water or buffer should be zero or very close. All measurements are automatically normalized to 340nm.
5. Measure the nucleic acid sample by loading 1microliter of sample and selecting "measure". Record your DNA concentration in your lab notebook and on the tube of plamid DNA. Once the measurement is complete. Clean both optical surfaces with a Kimwipe and the machine is ready for the next sample.
You should ensure that the appropriate constant (50 for dsDNA or 40 for RNA) has been chosen. The software automatically calculates the nucleic acid concentration. If the calculation is done by hand, the A260nm is represented as a 1cm path for convenience, even though 1-nm and 0.2nm paths are actually used during the measurement cycle.
When the last sample was been measured, clean the sampling device by repeating step 1.
Part 3: Transformation of plasmid DNA into E. coli strain HT115(DE3)
The competent HT115(DE3) bacterial cells are on the instructor’s bench. You will transform some of your plasmid DNA into these bacteria. The cells are very fragile, so treat them gently.
The genotype of the HT115(DE3) cells is: F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAse III minus). This strain grows on LB or 2XYT plates. This strain is tetracycline resistant. The most important things about this strain is:
- It has an IPTG inducible T7 polymerase. The chemical IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a lactose mimic and is commonly used to induce the production of large amounts of protein. In this case when the cells are incubated with IPTG lots of the polymerase T7 is made. This T7 polymerase binds to T7 specific promoters on the plasmid and transcribes lots of our gene of interest.
- RNAse III minus. The strain is lacking the RNAse III enzyme which typically breaks down double stranded RNAs. We want double stranded RNAs to accumulate.
The prep staff prepared these competent HT115(DE3) cells for you using the Inoue Method Media:Inoue bacterial transformation.doc. Reference: Inoue H., Nojima H., and Okayama H. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23-28. The cells were made competent to take up free plasmid DNA by this treatment. This treatment makes the cell wall and membrane more permeable and our transformation efficiency much greater but it weakens the cells; therefore you must keep them cold and mix them gently throughout the transformation (no vortexing!).
- Obtain a tube containing 50 μL of competent cells from your instructor.
- Label the top or the side of the tube with HT115(DE3), pPD12936+lsy-2, and your initials or team color.
- Consult with your instructor to determine how much of your plasmid DNA you should add to the cells to start the transformation. When you have determined the correct vol., pipet between 1μL and 10μL of your plasmid DNA to the tube. (The volume you should use is dependent on the conc. you achieved in your mini-prep.) Pipet up and down once to mix the DNA and the cells. Close the cap and let the transformation mixture sit on ice for 10 minutes.
- Heat shock by incubating the transformation mix at 42°C for 90 seconds, exactly. This step must be timed exactly. Remove the tube at the end of 90 seconds to your ice bucket while you get your LB ready.
- Consult your instructor to determine the volume of warm LB broth you should add (usually between 250-500 microliters) to the transformation mix. LB aliquots should be found in the 37°C mixer/incubator on the green/blue team bench. When pipetting the media, remember to release your thumb on your micropipet slowly, to avoid splashing the liquid on the end of the barrel. The barrel is not sterile and if you see the liquid touch it, then discard the media in the waste beaker and try again with a new tip.
- Once you have added the LB, close the cap and invert the tube once or twice to mix the contents. Incubate at 37°C for 30-45 minutes in the bench top incubator/ mixer with occasional gentle mixing.
- While the plasmid DNA is being taken up by the competent cells and the new genes provided by the plasmid are being expressed by the bacteria, label two LB + amp agar plates. Label the bottom of the plated with the strain's identity (HT115(DE3)), the plasmid used, the date, your initials and team color. You must label the bottom of the plated since the tops are easily switched. Differentiate them by putting 50μL on one and 200μL on the other. Put these plates in the hood with the blower on and with the lid ajar to dry the surface of the agar for about 10 minutes or until the surface looks dry but is not badly dehydrated.
- Once the transformation mix has incubated at 37°C for 30-45 minutes, invert it to mix the contents and pipet 50 microliters of transformed cells onto the center of a labeled slightly dehydrated LB + amp plate prepared in the previous step. Pipet 200μL on the other plate. Pour 5-10 glass beads onto the plates. Put the lid back on and gently swirl the beads all over the plates to spread the transformed bacteria around. When you are done - pour the beads into the beaker with disinfectant near the sink.
- Replace the lids and leave the agar plates undisturbed for a few minutes.
- Once they have dried enough that the surface doesn’t appear wet, invert the plates and incubate them at 37°C for 24-48 hours. The plates should be incubated with the agar side up so that condensation will not drip onto the surface of the agar and smear the colonies that will be growing there.
- Save the remaining transformation mix for 24 hours or until we are sure that there is at least one colony growing on each of your plates.
What would it mean if you had no colonies on your plate? Normally, you would expect to have around 100 pale color colonies on each plate. If you have at least one well isolated colony on the plate, you’re all set. After the 24 hour growth period the plate should be placed in the rack in the refrigerator labeled with your lab day. You will use a single colony from the plate to make an overnight broth culture on the day before next lab. If you have no colonies on one or more of your plates, please notify your instructor right away.
Before leaving lab today, give the rest of your isolated plasmid DNA to your instructor in a labeled microfuge tube. Make sure your tube is labeled with your name, lab day, plasmid name, DNA concentration and color coded with a piece of tape in your team color.
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 bacteria containing 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. Select a colony to start your overnight culture. At this point, ALL of your colonies should contain your plasmid of interest.
- Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the refrigerator in the back left hand corner of the room.
- Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator with the broth) to each tube. Calculate the effective concentration of ampicillin that you have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook.
- Add 5 microliters of the 12.5mg/ml tetracycline stock (also found in the refrigerator with the broth). Calculate the effective concentration of tetracycline that you havein your LB broth tube. Record that info in your lab notebook.
- Gently swirl your LB +amp+tet broth to mix the contents.
- Label the two sterile glass culture tubes with tape in your team color. Label one with "pPD129.36 lsy-2" and your initials. Label the other with your initials only.
- Inoculate the broth with your bacteria by using a sterile disposable loop to scrape your candidate colony off the plate. Be sure not to touch the plate with the loop except on the desired colony and don’t pick up any satellite colonies! Gently swirl the loop in the LB+amp+tet broth - you should be able to see the colony come off the loop. (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.) If you prefer to use a sterile toothpick rather than a loop, you may pick up the colony with the sterile toothpick and drop the toothpick into the broth culture. Note that the tip with the colony is in the broth and the contaminated part you touched with your fingers does not touch the sterile medium.
- Balance the 2 tubes across from each other on the rotating wheel in the 37C incubator at the front of the room when you come in the door. DO NOT USE THE ROOM TEMP WHEEL!
- Incubate these broth cultures at 37°C overnight. Do not forget to make sure the wheel is rotating when you leave!