BME103:T130 Group 14: Difference between revisions
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| [[Image:Photo_on_11-14-12_at_9.37_PM.jpg|120px|thumb|Name: Nathaniel Bennett<br>Role(s): PCR Technician/Research and Development Specialist]] | | [[Image:Photo_on_11-14-12_at_9.37_PM.jpg|120px|thumb|Name: Nathaniel Bennett<br>Role(s): PCR Technician/Research and Development Specialist]] | ||
| [[Image: | | [[Image:Brianh.jpg|100px|thumb|Name: Brian Hedden<br>Roles: ImageJ Software Processor/Data Compiler & Analyzer]] | ||
| [[Image: | | [[Image:BME103_Group 14.JPG|200px|thumb|Name: Hanna Rahman<br>Role: Protocol ]] | ||
| [[Image: | | [[Image:Me_bme.jpg|100px|thumb|Name: Hope Haddad<br>Role(s): Protocol/Research and Development Specialist]] | ||
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#Turn on the excitation light using the switch for the Blue LED. | #Turn on the excitation light using the switch for the Blue LED. | ||
# Place the smartphone on the cradle at a right angle from the slide. | # Place the smartphone on the cradle at a right angle from the slide. | ||
[[Image:Group14SmartPhone.JPG|400px|300px]] | |||
# Turn on the camera setting on the smartphone. '''Reminders:''' | # Turn on the camera setting on the smartphone. '''Reminders:''' | ||
## Turn off the flash. | ## Turn off the flash. | ||
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#Align the drop by moving the slide so that the blue LED light is focused by the drop to the middle of the black fiver optic fitting on the other sisde of the drop. | #Align the drop by moving the slide so that the blue LED light is focused by the drop to the middle of the black fiver optic fitting on the other sisde of the drop. | ||
#Cover the fluorimeter with the light box, but make sure you can access your smartphone to take the image. The light box should be used to remove as muhc stray light as possible. | #Cover the fluorimeter with the light box, but make sure you can access your smartphone to take the image. The light box should be used to remove as muhc stray light as possible. | ||
[[Image:Group14Box.JPG|400px|300px]] | |||
[[Image:Group14Covered.JPG|400px|300px]] | |||
#Take three images of the drop of the water. Make sure not to move your smartphone. | #Take three images of the drop of the water. Make sure not to move your smartphone. | ||
# Remove the box (make sure not to move your smartphone). If you need to adjust for any movement, use the ruler provided to measure the distance so that you can return to that location. You can also use ImageJ to compensate for moving the camera, but it makes the analysis more complicated. | # Remove the box (make sure not to move your smartphone). If you need to adjust for any movement, use the ruler provided to measure the distance so that you can return to that location. You can also use ImageJ to compensate for moving the camera, but it makes the analysis more complicated. | ||
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==Research and Development== | ==Research and Development== | ||
'''Specific Cancer Marker Detection - The Underlying Technology'''<br> | '''Specific Cancer Marker Detection - The Underlying Technology'''<br><br> | ||
'''Polymerase Chain Reaction'''<br> | |||
In order to replicate DNA to work on, we must copy strands of DNA through a process called DNA replication. Polymerase Chain Reaction(PCR) is used to determine a specific point on DNA that is to be copied. In DNA replication, a bubble of DNA primers and fluorescent tags will form around a specific piece of DNA and divide, or unzip (annealing) DNA. Any unneeded reagents in the solution will simply not bind to a base pair. In order for this to occur, the solution is heated to 95 degrees Celsius for about 3 minutes. Here, when the molecule is split into two, polymerase will add corresponding base pairs creating a first copy. For this to occur, the temperature needs to be brought down to about 57 degrees Celsius. At the same moment, polymerase is creating base pairs for the other half of the original DNA strand. The thermal cycler heats and cools the solution to complete the process. This process will cycle a few times in order to create duplicate DNA that is able to be tested for different reasons such as cancer. <br> | |||
[[Image:PCR_Group14.jpg|400px]]<br>(Image taken from biology.kenyon.edu)<br><br> | |||
The dye we added to each sample will fluoresce under UV light when DNA is present. If our eyesight was good enough, we would see that all the samples fluoresce slightly, however, because that is not the case, we do not. Instead, what we see fluoresce are samples that have a large amount of DNA present. We know from the experiment we created that we replicated cancerous DNA, specifically the r17879961 cancer associated sequence, meaning there would be excess amounts of the DNA in the sample if the patient has this cancerous sequence. Therefore, if a sample fluoresces bright enough for the naked eye to see, an excess amount of DNA is present and the patient does have cancer. | |||
<br><br> | |||
'''Bayes Rule'''<br> | |||
[[Image:Bayes-rule.png|200px]]<br> | |||
The Bayes Theorem allows a scientist to interpret new data based on their expectations. The calculations show to false positives and inconsistencies in data. This is important in experiments such as the probability of a patient having cancer through a PCR testing such as this. <br> | |||
Pr (A/B): Probability of a positive outcome | |||
P (B/A): Probability of a true positive | |||
P (A): Chance of having the cancer | |||
Latest revision as of 16:16, 15 November 2012
BME 103 Fall 2012 | Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OUR TEAMLAB 1 WRITE-UPInitial Machine TestingThe Open PCR machine is fairly easy to use. Simply plug it in to the USB port on your computer and turn it on. From your computer, you can adjust the temperature, add or remove steps to the process, and adjust the number of cycles.
When we unplugged the LCD from the Circuit Board, the LCD turned off. When we unplugged the white wire that connects the circuit board to heat block, the LCD was unable to accurately read the temperature.
The very first time that our group used the Open PCR machine was October 28, 2012. The process went fairly smoothly, which was unexpected. All we did was put the samples into the machine, set the peak temperature and number of cycles, then let it run for an hour and forty-nine minutes.
ProtocolsPolymerase Chain Reaction The polymerase chain reaction (PCR) is a biochemical machine used in biological chemistry to produce numerous copies of a particular piece of DNA, generating multiple duplicates of DNA sequences. The PCR machine works similar the cycle of DNA replication at the cellular level. The machine consists of four individual steps, initiation, denaturation, annealing, and extension. The initiation step is solely to prepare the DNA samples to be put through the thermal cycler program. During the denaturation step, the DNA strand is split into two separate strands. After, the annealing step is where the DNA primer attaches to the targeted DNA sequence. The primer only attached to a specific site on the strand, not necessarily the entire strand. The purpose of the primer is to mark the beginning and the end of the targeted DNA sequence. Lastly, in the extension step, the DNA polymerase is first activated, which begins to synthesize the DNA primer. This results in two double stranded target DNA sequences. The PCR machine cycles numerous times to amplify the specific sequence. In order to complete the reaction several components are required such as:
Table 1
Table 2
Table 3
Flourimeter Measurements
Type of smartphone used: Iphone 4s In this lab, we used SYBR Green I, which is used to detect a particular DNA strand. In this lab, our fluorimeter was created using optical caustic because it eliminated the need for lasers, mirrors, and lenses. A fluorimeter is a device used to measure parameters of fluorescence: it's intensity and and wavelength distribution. The quality is related to the amount of florescent material and indirectly proportional to the molecule being detected. The objective of this part is to determine if you have actually amplified the target DNA in your PCR experiment. Through this you will be able to visually observe the fragments and calculate the relative amount of DNA. During the first procedure, you amplified your DNA, so now that you are going dye it with the following procedure:
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology
Results
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