Cybertory™ Virtual Molecular Biology Laboratory
PCR Detection of Toxic Escherichia coli
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Summary
In this exercise, we will use Polymerase Chain Reaction (PCR) to amplify a sequence unique to the toxic O157H7 strain of E. coli bacteria. PCR requires primers, template, polymerase, buffer, and dNTPs; each component must be present in appropriate concentrations, or the reaction will fail, leading to false negative results. PCR is also very sensitive to cross-contamination, which can lead to false positives. Successful completion of this exercise requires carefully following a protocol over a large number of pipetting steps. Positive and negative controls are essential for interpreting the results, and for troubleshooting possible technical problems.
Objectives
- Learn to follow protocols to assemble complex in vitro reactions by pipetting ingredients into reaction tubes.
- Understand the importance and design of positive and negative control reactions, both for interpreting results and for troubleshooting technical problems.
- Practice preventing cross-contamination of samples, primarily through using disposable pipette tips.
- Understand how PCR can be used to test for microbial contamination.
Background
E. coli are a gram-negative rod-shaped bacteria commonly found in the human intestinal tract. Detecting this species in environmental samples usually indicates contamination with sewage. Most strains are harmless, but some strains are toxic and can cause disease. The most important of these toxic strains is O157 (that's the letter 'O' and the number 157). It has been found in contaminated hamburger, where it has caused outbreaks of bloody diarrhea.
In this exercise, you will use the Polymerase Chain Reaction (PCR) to detect the toxic strain. We do not want to recall hamburger that only has harmless bacteria, so you will also evaluate this detection method on normal E. coli, to be sure it only identifies the toxic strain.
If you are not careful, particularly about changing pipette tips, you can cross-contaminate your samples, so that samples which should be negative appear positive. A major reason to run negative controls is to detect cross-contamination making everything positive.
You need to be aware that there are a lot of things that can go wrong with PCR reactions in the Virtual Laboratory. The polymerase enzyme is sensitive to pH, dNTP concentrations, and Mg++ concentrations, and reactions can fail if these conditions are not correct, within very narrow margins. The positive control reactions are very important, because we need to be able to tell the difference between a negative result (where we do not see a PCR product because the sample being tested does not contain the target sequence) and a non-result (where we do not see bands because the reaction failed.) The positive control for our template will be DNA from an E. coli O157H7 strain, and the negative control will be from the non-toxic E. coli strain K12.
We will run three experiments in this exercise, each a bit more complicated than the one before. First, we will do PCR with control reactions in tubes that have already been mixed together, and run them on a gel to be sure everything is working. Second, we will mix up four control reactions ourselves, to make sure we get the same results. Finally, we will mix up a whole set of 12 reactions, and compare the results of four 'unknown' samples to our positive and negative controls. For the larger number of reactions, we will save a number of pipetting steps by using a 'master mix', where we assemble the common ingredients for a set of reactions in a single tube.
Equipment
Read the user interface section of the FAQ for clues on how to use the Virtual Lab.
Below is a list of the various pieces of furniture and equipment used in this experiment. Each item links to its entry in the Simzymes catalog, where you may find further information about that item and how to use it.
Protocol
You may want to print this protocol so you don't have to keep flipping back and forth between windows.
- In the drawer beneath the PCR machine, you will find a tube rack with four tubes, labelled "rK", "rO", "tK", and "tO". These are control reactions using known templates: the first letter indicates the primers used ('r' for 16 rRNA primers that should work on all strains of bacteria, and 't' for the gene unique to toxic E. coli O157H7), and the second indicates the bacterial template (two strains of E. coli:'K' is K12, and 'O' is O157H7).
- Place the four samples in the PCR machine, and press the "go" button. Fast-forward the clock to speed up the machine (note that there are several different fast forward speeds; just you click the clock control multiple times).
- Load the PCR products on a gel by pipetting 15 µl from each sample into consecutive wells of the gel:
- rK
- rO
- marker (100 bp ladder, in the drawer below the gel box)
- tK
- tO
- Turn on the electricity by pressing the 'power' button on the power supply. Fast forward the clock to speed up the gel.
- In the cabinet below the gel box is a camera hood. Click the button on the camera to set the type of file you want, and drag and drop the camera hood onto the gel box to take a picture. Drag it away and drop it anywhere not touching the gel box to have it put itself away.
Run pre-made controls
- Refresh the lab window, so you start with a new gel and all fresh reagents. Now set up the same four control reactions yourself as follows:
- Pull four new tubes from the tube jar and place them in the bottom right rack. Put two in the first row, and label them 'rK' and 'rO', using the felt marker (in the second drawer). Put the other two in the second row, and label them 'tK' and 'tO'.
- Examine the contents of the ice bucket and the four tube racks on the bench top of the first bench unit. You can either zoom in to read the labels, or hover the mouse cursor over each tube for a few seconds until the magnifying glass pops up. It is important to note the layout of the tubes because it can be hard to read the labels when you are zoomed out enough for mixing together the reactions. The ice bucket contains a tube of Taq polymerase and one with dNTPs. The rack just below the ice bucket has water and a tube of 10X buffer. The rack on the lower left has two pairs of primers: the pair on the top row (BAC16S-F and BAC16S-R) amplify ribosomal 16S RNA genes, and the pair on the bottom row (O157-FHC1 and O157-FHC2) amplify the sequence specific to O157H7. The rack just below the tip box and pipette stand has six template solutions. The first two are controls templates of known bacteria, and the other four are samples to be tested. You will be mixing your reactions using new tubes in the empty tube rack at the lower left. You should set them up so that each column contains the same templates as in the template rack, and each row contains the same primers as in the primer rack. The reactions will use these primers and templates:
Tube Primers Template rK BAC16S-F and BAC16S-R EcoK12 rO BAC16S-F and BAC16S-R EcoO157H7 tK O157-FHC1 and O157-FHC2 EcoK12 tO O157-FHC1 and O157-FHC2 EcoO157H7 - Each reaction should have the following ingredients, where the primers and template are taken from the list above:
Be sure to put a clean tip on the micropipette each time it is used with any solution that contains DNA.Ingredient Volume H2O 10 µl 10X Taq Polymerase buffer 2 µl Taq polymerase 1 µl dNTPs (10X stock is 2mM each) 2 µl primer A 2 µl primer B 2 µl template 1 µl - Run these control reactions on a gel, as you did earlier with the pre-mixed controls. If you do not get the same result, try to figure out what went wrong.
Mix your own controls
- Now that you have some experience in setting up PCRs, you can run the full set of reactions. Reload the laboratory so that you get a fresh gel, and all new reagents.
- Put twelve clean tubes in the empty rack. As we did with the controls, we will put one template per column, and one pair of primers per row.
- Make one master mix per row of reaction tubes. Since the mixing rack is full, use the one to its left (the rack with the primers) to hold the master mixes. Put new tubes in the rightmost two positions of that rack.
- Each master mix needs to hold enough solution to make six reactions. You need to make enough for an extra reaction to account for possible pipetting loss. Assemble the following ingredients into each master mix tube:
Ingredient per rxn Master 10X Taq Polymerase buffer 2 µl 14 µl primer A 2 µl 14 µl primer B 2 µl 14 µl Taq polymerase 1 µl 7 µl dNTPs, 2mM each 2 µl 14 µl H2O (to 19 µl) 10 µl 70 µl - Pipette 19 µl into each reaction tube from the appropriate master mix.
- Pipette 1 µl of template into each reaction tube from the corresponding column in the template rack.
- Run the PCR, load the gel, and take a picture as before.
Test unknowns
Discussion Topics
- Which unknown samples contain E. coli O157H7?
- Can you devise an experiment to determine how much you can dilute the positive control template and still produce visible bands?
- Why is the possibility of sample contamination particularly important in PCR?
- How might using a master mix make your negative controls more informative?
- What aspects of the virtual lab are different from what you would expect in a real lab?
References
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