Once the student launches the Molecular Biology laboratory, the student is placed in front of a lab bench containing a large assortment of experimental equipment required for performing basic molecular biology experiments. The overall goal of the laboratory is to give students the opportunity to produce DNA sequences of amplified segments and genes and then compare those sequences among related species.
To use the laboratory, students can select up to eight microcentrifuge tubes and place them in a tube holder. Students can then select species using the species menu and for each selected species add a small DNA sample to any of the tubes in the tube holder using the micropipet. In addition to the DNA samples, students can also add Taq polymerase (Taq), nucleotides (dNTPs) or fluorescently tagged nucleotides (dNTPs tag), and selected primers to the tubes in preparation for amplification of a selected gene or segment by PCR. Once the tubes have been prepared they can be moved to the PCR machine where the appropriate denaturation, primer annealing, and elongation temperature and dwell times can be configured for a PCR experiment. After the PCR run, samples of the amplified products can be placed in a well on an electrophoresis gel bed and a voltage applied to separate the products into bands to determine the approximate mass of the products. Once a successful amplification of the selected gene has been confirmed, then each amplified product can be placed in the automatic sequencer and the amplified gene sequence determined. Measured gene sequences can be saved to the lab book for further analysis and comparison with other sequences. The essential features of the Molecular Biology laboratory are described below starting on the left side of the lab bench and moving to the right.
Lab Book. The lab book menu is used to record procedures and observations while performing experiments in the virtual laboratory. Gene sequences that have been measured in the laboratory can also be saved in the lab book where they can be viewed and copied to analyze in other software. The results from gel experiments can also be saved to the lab book. See the Lab Book help menu for further details about using the lab book.
Pipet. The pipet is used to transfer DNA, primers, and reagents to the tubes or to transfer samples from tube to tube or to the gel. The species menu and primer menu each have pipet icons that are used to fill the pipet with DNA of the selected species or with the selected primer. The pipet can also be picked up by clicking once on the pipet on the lab bench. Using the pipet is a matter of dragging the pipet over a tube, bottle, or well and clicking. If the pipet leans to the right then the pipet is empty and clicking fills the pipet. If the pipet leans to the left then the pipet is full and clicking empties the pipet. Clicking anywhere in the lab (except the bottles or tubes) will place the pipet back on the lab bench.
Bottles. On the back of the lab bench are bottles of Taq polymerase, untagged nucleotides (dNTPs), and fluorescently tagged nucleotides (dNTPs tag). The Taq and nucleotides are necessary reagents for a PCR experiment and are added to the tubes using the pipet. Note that the tagged nucleotides are necessary for the sequencer reaction to determine the gene sequences, but the tagged nucleotides are more expensive than the untagged.
Tube Rack. The tube rack contains slots for 8 different tubes. Tubes can be placed in any of the slots from the Tube Jar or moved from the PCR machine. A label can be attached to a tube by hovering directly below the selected tube, clicking in the label area, and then typing in a label. Hovering over the tube shows the label on the LCD screen on the rack and as a popup inside the PCR machine.
Primers. The primer menu contains a list of available primers that can be added to the sample tubes. The primer list contains all primers that are necessary to amplify the available genes and also a random set of primers that are not needed. Custom primers can also be defined by typing in a customer primer sequence. Primers are added to the sample tubes by selecting a primer, clicking on the pipet icon, and then dragging the pipet to the desired sample tube and clicking to add the sample. Two primers are required for a successful gene amplification.
Tube Jar. The tube jar contains an endless supply of microcentrifuge tubes needed for the experiments. New tubes are obtained by hovering over the tube jar to take off the lid and then clicking and dragging a new tube to the tube rack or the PCR machine.
Laboratory Clock. When the molecular simulation is first started, the laboratory clock shows the current time of day in a 24-hour format. Many of the experiments in the laboratory are very time consuming and it is not necessarily realistic to wait in real time for these experiments to finish. For example, a PCR experiment can take over 3 hours and a sequencer run can take anywhere from 3 to 15 hours. Consequently, the laboratory clock can also be used to advance the laboratory time to accelerate these experiments. Time is advanced by clicking on any of the arrows above each digit on the clock.
PCR Machine. The PCR machine takes up to 8 tubes of DNA samples and runs them through a three step temperature and hold profile for a specified number of cycles. If the temperatures and hold times, primers, Taq, and nucleotides have all been added appropriately, then the number of target genes or strands will be doubled at the end of each cycle. The three steps in each cycle are Denaturation, Primer Annealing, and Elongation where each step occurs at a specific temperature for specific hold time. Given here is a brief description of each element of the PCR machine.
- LCD Panel. The LCD panel displays status information about the current PCR run. The top line shows the current
temperature and time remaining for the current step, the second line shows the number of cycles that have been completed, and the last line displays the current PCR operation. - Control Panel. The control panel consists of the green Start button and the red Cancel button. These buttons are used to start and stop a PCR run.
- Lid. The lid on the PCR machine must be opened before adding sample tubes. The lid is opened and closed by clicking on the lid.
- PCR Controls in Setup and Live Data menus. The PCR section in the Setup menu is used to specify the temperatures and dwell times for each of the three PCR steps. The Live Data PCR section can also be used to start, stop, and reset
the experiment, and displays an animation of the PCR process at the molecular scale while the PCR experiment is running.
Nucleotide and Amino Acid Charts. The nucleotide and amino acid charts on the wall show the chemical structures of the five base pairs (A, T, G, C, and U), the twenty amino acids, and the base pair sequences that produce each amino acid. Clicking on the chart will expand the chart for easier viewing.
Gel Electrophoresis. In the gel electrophoresis experiment, amplified product is pipetted into one of 8 different wells in the
gel and a voltage is applied across the gel bed to move the samples through the gel and separate any DNA mixtures into bands according to their mass or number of base pairs. Heavier bands are always at the top of the gel since they move slowest when a voltage is applied. Samples are added to the wells on the gel using the pipet to transfer amplified product from a tube, dragging it across the gel to identify the well, and then clicking once to pipet the sample into the well. Students must attach the voltage leads to the gel bed as part of the gel experiment. This is done by clicking and dragging each lead to the appropriate location on the top or bottom of the bed. The power supply below the lab bench is used to control the gel experiment.
The gel electrophoresis display in Live Data is used to view the appearance of the gel bed before, during, and after the experiment. The display depicts the gel in room light with the wells at the top. The first and last lanes on the gel are used for a standard set of markers that are 200, 400, 800, 1000, and 2000 base pairs. Bromophenol blue is used as a tracking dye in each lane to track the movement of the bands, and SYBR is used as a marker dye to view the position of each band with UV light.
Across the top of the gel display are several buttons. Save is used to save the gel image to the lab book. This button is only active when the UV light is on. The UV button turns on the UV light to expose the marker dye. When the UV light is on a Pipet icon will also be available. This icon is used to pick up the pipet and extract a DNA sample from the separated bands for further amplification. Lastly, on the right is a Horizontal Tool that can be used to measure or estimate the mass of any bands on the gel. This is done by clicking on the icon and then dragging the horizontal tool down the gel. The right hand side of the gel is used to display the estimated mass.
Power Supply. The power supply is used to control the voltage and the time the voltage is
applied to the gel experiment. The main power button is located next to the voltage leads, and the LCD screen allows you to set the duration, the voltage, and displays the time remaining on the experiment. The On/Off button is used to actually apply the voltage to the gel, and the voltage will turn off automatically after the specified time has elapsed.
Sequencer. Sequencers are used to determine the DNA sequence of the amplified product. Actual DNA sequencers are sophisticated pieces of equipment and require a unique set of reagents. The virtual automatic sequencer used in the molecular laboratory is completely automated and requires only that the student has amplified enough product to provide a sufficient quantity for the sequencer reactions. A sample is loaded into the sequencer by clicking on and dragging a sample tube over to the sequencer drawer, holding over the drawer until it opens, and then dropping the tube on to the tube holder. Clicking on the drawer closes it and initializes the instrument for a sequencer run.
The sequencer controls are in Live Data. The sequencer section displays the results of the sequencer run in real time. Clicking on the Start button starts a sequencer run and it can be stopped at any time by clicking on the Stop button, although the run will automatically terminate when the end of the sequence has been reached. Note that the sequencer produces a base pair every 30 seconds so it may appear that the sequencer is not working. It will be convenient to use the laboratory clock to advance the time since sequencer runs can take many hours.
After one or more sequences have been determined and are displayed in the sequencer window, other functions are also available. A sequence is selected by clicking on the sequence, which then activates the Save button and allows the student to
enter a name for the sequence. Clicking on the Save button saves the sequence to the lab book for further analysis. The lab book icon is used to indicate if the sequence has been saved to the lab book. An alignment tool is also available in the upper right corner of the window. This tool can be used to check for similarities in the sequence for a list of sequences. The Zoom button opens up a window with the sequences to allow for comparison and analysis of the sequences.
Presets. The list of Lab Presets is a set of preset molecular biology experiments that are already predefined and ready to run. Clicking on the preset will clear the laboratory and launch the preset.