BME103:T130 Group 13
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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LAB 1 WRITE-UP
When we unplugged the PCB Board of the LCD from the Open PCR Circuit Board, the machine's LCD screen turned off.
When we unplugged the white wire that connects the Open PCR Circuit Board to the 16 Tube PCR Block, the machine could not register or measure the temperature.
On October 18, 2012, our group first tested the Open PCR Machine. At first, the machine seemed overwhelming in its design. However, after following the instructions and advice from peers and professors, we were able to determine how to properly setup, program, and run a simple test.
Polymerase Chain Reaction
Polymerase Chain Reaction is a technology that amplifies a single piece of DNA. This technology works very similarly to the natural DNA replication cycle. One PCR cycle consists of three basic steps, denaturation, annealing and extension. In the denaturation step, heat (usually about 95 degrees Celsius) is used to separate the DNA into two strands. Then in the annealing step, the temperature is decreased to 50 degrees Celsius and the DNA primer, specific to the target sequence for that organism, anneal to the separated strand of DNA. The primers mark the beginning and the end of the targeted DNA sequence. Finally, the extension step required the temperature to be raised to 72 degrees Celsius so that the DNA polymerase is activated. The DNA polymerase begins synthesis at the DNA primer. This results in two double stranded target DNA sequences. The PCR cycle is repeated many times to amplify the targeted strand.
There are typically many cycles that need to take place in the PCR in order to amplify a patient's DNA.
|Template DNA (20ng)||0.2 μL|
|10 μM forward primer||1.0 μL|
|10 μL reverse primer||1.0 μL|
|GoTaq master mix||50.0 μL|
|Total Volume||100.0 μL|
We ran a total of 8 samples. We made 3 samples from Patient 1 (ID#65685) was female and aged 58. We made 3 samples from Patient 2 (ID#58278) who was also female and aged 53. The remaining 2 samples were the positive and negative controls. The positive control for this experiment was the cancer DNA template and the negative control was the no DNA template.
Fluorimeter Assembly Procedure
1) Turn on the excitation light using the switch for the blue LED.
2) Place a smart phone on the cradle at a right angle from the slide.
3) Adjust your camera settings as follows:turn off the flash, set the ISO to 800 or higher, increase the exposure to the maximum, and turn off autofocus (optional).
4) Move the smartphone in the cradle as close possible to the first two rows of the slide so that you will get a clear image.
5) The pipette should be filled with liquid only to the bottom of the black line. Then use the pipette to place two drops of water (each drop should be between 130-160 microliters) in the middles of the first two rows of the slide.
6) Move the slide so that the blue LED light is focused on the the drops of water to the middle of the black fiber optic fitting on the other side of the drop.
7) Cover the fluorimeter with the light box so that much of the stray light will removed, but make sure you can still access your smartphone to take pictures.
8) While being careful not to move the smartphone, take three picture of the water droplet.
9) When removing the light box, be careful not to move the smartphone because that could make the analysis more complicated.
10) Use a clean plastic pipette to remove the water droplets from the slide.
11) Push the slide in so that you are npo in the next set of two holes.
12) Repeat steps 5-10 four more times in 5 different positions.
13) Record the following: type of smartphone used, the distance from the base of the smartphone cradle to the measurement device (in cm), and attach one image for each position of the drop (5 images total).
Transferring the images from your smartphone to the laptop that has ImageJ
1) Connect your smartphone to your laptop using a USB sync cable.
2) Click Start and then click My Computer or Computer where under Portable Devices, you should find your smartphone icon and double-click on this icon.
3) Once you have opened it, double click folder DCIM, and next double-click the folder Camera.
4) From the Camera folder, press down on CTRL and click on the images you want to transfer and right-click and copy these images. NOTE: Do not take your finger off the CTRL key until after you right-click.
5) Under the Libraries folder, click on the Pictures tab and right-click and go to New and select Folder. Name this new folder ImageJ Pictures and double click on this folder. You can now right-click and paste the images taken by your smartphone into this folder.
6) Go to your desktop and double-click on the ImageJ icon and when ImageJ opens, go to the top left of the bar and click on File, next click Open.
7) A folder will appear on your screen, and on the left click the Libraries icon, next double-click the Pictures icon. In the Pictures folder, find the ImageJ Pictures folder you previously created and double-click on that folder.
8) In the ImageJ Picture folder, select an image (you can only select one image at a time) and click Open. In a few seconds, the image will be appear on your screen.
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
The reason that the cancer-associated sequence of r17879961 will produce a DNA signal while the non-cancer DNA sequence of the same SNP (single nucleotide polymorphism) will not produce a DNA signal lies in the arrangement of nucleotides at the molecular level. The lack of a DNA signal is due to the inability of the reverse primer to bind to the forward strand during the annealing phase of PCR. To detect the cancer-associated sequence of r17879961, the reverse primer AAC TCT TAC ACT CGA TAC AT is used. This is because the cancer-associated mutation is represented by a single nucleotide in a particular triplet: instead of the normal ATT, the middle T mutates into a C, thus rendering a triplet of ACT (which you can see in the reverse primer shown above). At the protein level, this mutation of 1 nucleotide changes the coded protein from isoleucine to threonine. As a result, the primer will not attach to the normal r17879961 DNA sequence as it will not have the corresponding base pairs (TGA) in the particular section of DNA that the mutated sequence would have.
(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)
(Your group will add the results of your Fluorimeter measurements from Week 4 here)