BME103:T130 Group 10
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
Experimenting With the Connections
When the circuit board (part 6) was unplugged from the display (part 3), the display turned off and no longer registered any signal. The display was no longer functional, but turned on again once the wire between the two parts was reconnected.
When the circuit board was unplugged from the heating element, the heating element was no longer able to control its temperature. Although it still had power, it was not being controlled by the circuit board.
The first test run completed on the OpenPCR machine was on October 25, 2012. The test was successful, and was completed in roughly the time expected, give or take a few minutes. Once the settings were put to the correct levels, the set up was quite simple. The samples were placed inside the heating unit, then the test run started. It took over an hour, but was completed successfully with no malfunctioning pieces of equipment.
Polymerase Chain Reaction
1.)HOW PCR WORKS
The Polymerase Chain Reaction machine, PCRm for short, works by cycling DNA through different temperatures to amplify the desired strand so the sample can be compared with other DNA. First, the DNA is denatured by heating the samples to such temperature that the hydrogen bonds holding the double-stranded molecule together are broken. The sample is then cooled, which allows a primer to bind to the target DNA. In the third step, the sample is heated back up so an enzyme can replicate the DNA. In the final step, a fluorescent dye binds to the new double-stranded DNA. This process is repeated many times, amplifying the target strand to an analyzable quantity.
2.)STEPS TO AMPLIFY DNA
The actual steps for PCR are quite simple.
Step 1. The PCRm lid is heated to 100°C and the sample tubes are heated to 95°C. This temperature is held constant for 3 minutes.
Step 2. The PCRm is then set to run 30 consecutive temperature cycles. Each cycle consists of heating the sample to 95°C for 30 seconds, cooling to 57°C for 30 seconds, and finally, heating to 72°C for 30 seconds.
Step 3. After the series of cycles is completed, the PCRm temperature is then held at 72°C for 3 minutes.
Step 4. The PCRm is then kept constant at 4°C
3.)COMPONENTS OF PCR MIX
4.)TABLE OF REAGENTS AND VOLUMES USED
1.)PHOTO OF SETUP
2.)STEPS TO ASSEMBLE THE FLUORIMETER
To assemble the fluorimeter, there are a few easy steps that need to be followed. The first step is to unbutton the front flaps and open the lid. Take out the interior contents which include the slide that the sample is put on and the cell phone stand. Once this is done, close the lid so that only the front is open. Place your sample on the slide, turn on the light and place the entire ensemble inside the box. Then put a cell phone with a camera in the cell phone stand. Align the camera with the sample on the slide. After this step, the fluorimeter is set up. Refer to the image above for a photo of the set up.
3.)USING IMAGE J
We used the program ImageJ to analyze our results. In ImageJ, we first opened the photograph taken using the fluorimeter setup. We then split the photograph into color channels (IMAGE -> COLOR -> SPLIT CHANNELS) in order to separate the "green" of the image from the overall photograph. The SYBRGreen's fluorescence can be analyzed with any expectation of validity only in isolation. The blue and red channel images were disgarded. Before actually measuring the image, we set the desired parameters of measurement (ANALYZE -> SET MEASUREMENTS -> AREA, INTEGRATED DENSITY, MEAN GRAY VALUE). Then, using the "oval" tool, we selected the sample solution in the image. After selecting the sample, we measured for the selected data (ANALYZE -> MEASURE) and recorded the results. Without changing the shape of the selected area, we dragged the oval to be in the background of the image and repeated the measurements. The background numbers are needed to acc