BME103:T130 Group 11: Difference between revisions
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* '''Integrated Density''' = This is the amount of pixels in an image or a selection of an image. We got these data by subtracting the integrated density of the drop selection and the black background. | * '''Integrated Density''' = This is the amount of pixels in an image or a selection of an image. We got these data by subtracting the integrated density of the drop selection and the black background. | ||
* '''DNA μg/mL''' = This was calculated by multiplying the Integrated density of the sample, with the background subtracted, by two and then dividing by the calf thymus integrated density. | * '''DNA μg/mL''' = This was calculated by multiplying the Integrated density of the sample, with the background subtracted, by two and then dividing by the calf thymus integrated density. | ||
* '''Conclusion''' = Positive means that the sample contains a positive test result cancer gene; no signal means | * '''Conclusion''' = Positive means that the sample contains a positive test result cancer gene; no signal means the sample contains a negative test result for the cancer gene. The samples that resulted with no signal meant that it produced a concentration that was smaller than that of the negative control. The samples that contained a positive test result produced a higher concentration than the negative control. From the data obtained, it appears that Patient 2 yielded a high level of DNA μg/mL, which resulted in a positive result for cancer in the first and second reps. In conclusion, Patient 2 most likely contains the cancer gene and Patient 1 does not based on the acquired results. | ||
Revision as of 17:02, 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 PCR machine heats up the DNA so that enzymes can "unzip" the two strands of DNA. This process happens in cycles so that the DNA will seperate and duplicate a multitude of times. A certain amount of primer is used to duplicate the DNA specific to the amount of original DNA. By amplifying the amount of DNA, a proper diagnosis of a certain gene can be made.
Experimenting With the Connections When we unplugged the LCD screen from the circuit board, the machine's screen shut off. When we unplugged the white wire that connects the circuit board to tube PCR block, the machine stopped reading the temperature. Test Run We first tested the PCR machine on the 18th of October, 2012. We initially ran our sample testing on the 1st of November, 2012 on PCR machine 12. The LCD screen readings matched the reading on the computer PCR program, and the machine worked well and efficiently.
ProtocolsPolymerase Chain Reaction (PCR)
The Polymerase Chain Reaction (PCR) is a process that depends on a DNA Polymerase enzyme's ability to synthesize a strand that is complementary to a targeted fragment of DNA in a test tube mixture of all four DNA bases, which are adenine, cytosine, guanine and thymine. Besides that, the test tube mixture also must have two fragments of DNA of about 20 base pairs that are called primers. These primers should have sequences complementary to adjacent areas of each side of the targeted DNA segment. Since these two primers should match exactly with only the targeted DNA sequence, only this area would be defined and copied.
First of all, the sample to be tested would be added to the GoTAQ PCR master mix. Secondly, the mixture would be heated to separate the sides of the double-stranded DNA. Thirdly, the mixture would be cooled to an optimum temperature for the primers to find and bind to whichever side of the separated DNA strands that they are complementary to. Lastly, the temperature is then raised slightly to reach an optimum temperature for the polymerase, which is included in the test tube mixture, to extend the primers so that new complementary strands are generated. At the end of the first cycle, there will be two copies of the targeted DNA segment. The cycle is repeated multiple times to generate more and more copies of the targeted DNA segments. With the advent of technology in biological sciences and engineering, the entire process can be automated after all the correct components are added into a tube by using a thermocycler or a PCR machine such as the OpenPCR machine.
The PCR master mix that was obtained from Promega consists of several different substances. These include nuclease-free water, deoxynucleotide triphosphates (dNTPs), magnesium chloride, and reaction buffers at optimal concentrations for any amplification of DNA. The reaction buffers had a pH of 8.5, and were made of 400μm deoxyadenosine triphosphate (dATP), 400μm deoxyguanosine triphosphate (dGTP), 400μm deoxycytidine triphosphate (dCTP), 400μm deoxythymidine triphosphate (dTTP) as well as 3mM of magnesium chloride.
This table below lists all of the reagents used as well as the volume used during this process.
Patient 1 ID #: 11640 Gender: Female Age: 54 years old
Patient 2 ID #: 29292 Gender: Male Age: 63 years old
The pipette with the blue strip was used to put two large drops of SYBR GREEN I on the first two centered drops on the glass slide in the fluorimeter.(Warning: please do not dispose of any of the pipettes used until the entire process is complete!) Once that was done, The corresponding pipette for the positive control was used to add two drops of the sample of the positive control to the two drops of the SYBR GREEN I that was already on the glass slide. The light in the fluorimeter was aligned to ensure that it was going through the drop. The fluorimeter was covered with the black file box and the smartphone operator was allowed to take as many pictures as needed. After the pictures were taken, the pipette with the black strip was used to dispose of the drop on the glass slide into the cup. The slide was then pushed forward so that the light would be in the general direction of the next two centered holes on the glass slide.
The above process was then repeated using the next samples available, which were the negative control sample, sample 1a, 1b, 1c, 2a, 2b and 2c. Once the first five samples were done, shifting the glass slide down two holes after every sample, that glass slide was disposed off, and a new glass slide was used.
Flourimeter MeasurementsTubes
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology PCR helps to detect certain types of genes. In this case it is used to find out a specific type of cancer. In the process of detecting the cancer, primers are made to compliment a DNA strand that has the cancer gene in it. If a subject has the cancer in their DNA, the primers will bind to strand, whereas a subject without the cancer would not have a primer attach to their DNA strand. The r1787996 SNP is linked to the cancer sequence. The codon ATC is the sequence for cancer where the ATT means there is no cancer. In PCR, the ATC cancer sequence is detected because the primers will only attach to the DNA strands that have the ATC sequence. The ATT, non-cancer, strands will not bind with the primers. Only the combined primer DNA strand will be detected thus alerting for cancer.
Bayes' Theorem
This rule can be used to determine the association between the probability of testing for cancer and whether or not the patient actually has cancer
Results
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