BME103:T130 Group 6: Difference between revisions
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'''The Original Design'''<br> | '''The Original Design'''<br> | ||
[[Image:Open PCR Solid Works Design.png]] | [[Image:Open PCR Solid Works Design.png|450px]] | ||
[[Image:BME 103 PCR SW.png]] | [[Image:BME 103 PCR SW.png|450px]] | ||
The images above depict our OpenPCR machine. It performs polymerase chain reactions (PCR), a process in which a particular DNA sequence is amplified. The DNA sequence can then be easily analyzed. The OpenPCR machine accomplishes the amplification of a DNA sequence through a series of heating and cooling sequences. <br> | The images above depict our OpenPCR machine. It performs polymerase chain reactions (PCR), a process in which a particular DNA sequence is amplified. The DNA sequence can then be easily analyzed. The OpenPCR machine accomplishes the amplification of a DNA sequence through a series of heating and cooling sequences. <br> | ||
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<b>1.</b> The Polymerase Chain Reaction or PCR works by singling out a single piece of DNA and then multiplying it so there's millions of copies of one strand of DNA. It's a step by step process that first occurs by heating up the DNA to 100°C in order to denature the hydrogen bonds between the two strands of DNA so that both sides of the DNA can be accesible to copy. After the strands are separated, specific primers are added to locate the section of DNA to be amplified. Next, the Taq DNA polymerase is added which actually copies the section of DNA desired and synthesizes the second half of each strand. After this there are only a few copies of the DNA which is why the machine then replicates more strands by repeating the process multiple times until there are millions of copies. <br> | <b>1.</b> The Polymerase Chain Reaction or PCR works by singling out a single piece of DNA and then multiplying it so there's millions of copies of one strand of DNA. It's a step by step process that first occurs by heating up the DNA to 100°C in order to denature the hydrogen bonds between the two strands of DNA so that both sides of the DNA can be accesible to copy. After the strands are separated, specific primers are added to locate the section of DNA to be amplified. Next, the Taq DNA polymerase is added which actually copies the section of DNA desired and synthesizes the second half of each strand. After this there are only a few copies of the DNA which is why the machine then replicates more strands by repeating the process multiple times until there are millions of copies. <br> | ||
<br> | <br> | ||
<b>2.</b> | <ul><b>2. Procedure</b> | ||
<li>1. Collect DNA from patients</li> | <li>1. Collect DNA from patients</li> | ||
<li>2. Heat Denaturation- the sample is heated to break the bonds between the strands</li> | <li>2. Mix the DNA samples with the GoTaq master mix and place into the PCR machine</li> | ||
<li> | <li>3. Heat Denaturation- the sample is heated to break the bonds between the strands</li> | ||
<li> | <li>4. Primer Annealing- the solution cools and the DNA matches up to the primer.</li> | ||
<li>5. Extension- The DNA replicates to produce millions of copies of the one strand of DNA .</li></ul> | |||
<br><br><b>3.</b> The GoTaq master mix contains 400µM dATP, 400µM dGTP, 400µM dCTP, 400µM dTTP, and 3mM MgCl<sub>2</sub> | <br><br><b>3.</b> The GoTaq master mix contains 400µM dATP, 400µM dGTP, 400µM dCTP, 400µM dTTP, and 3mM MgCl<sub>2</sub> | ||
<br><br><b>4.</b><b>Reagents used in our PCR test</b><br> | <br><br><b>4.</b><b>Reagents used in our PCR test</b><br> | ||
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'''Flourimeter Measurements'''<br> | '''Flourimeter Measurements'''<br> | ||
<br><br><center> | <br><br><center> | ||
[[Image: | [[Image:Fluorometer diagram.png|400px]]</center><br><br> | ||
<b>SET UP:</b><br> | <b>SET UP:</b><br> | ||
1. Take the file box and place it upside down (after it's been emptied) so that there is now an area that will restrict as much light as possible from coming through in the pictures.<br> | 1. Take the file box and place it upside down (after it's been emptied) so that there is now an area that will restrict as much light as possible from coming through in the pictures.<br> | ||
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<br> | <br> | ||
r17879961 is a sequence of DNA that has been positively linked with cancer. It is a part of a sequence of DNA that codes for a protein kinase called CHEK2. | r17879961 is a sequence of DNA that has been positively linked with cancer. It is a part of a sequence of DNA that codes for a protein kinase called CHEK2. | ||
<br><b> Bayes' Law Pr(A|B) = Pr(B|A)*Pr(A) / Pr(b) </b> | |||
<br> Pr is the probability, where A is an event and B is another event. The equation calculates the probability of an event happening if another event is true. | |||
<br><br> | <br><br> | ||
[[Image:PCR diagram.png|1000px]]<br> | [[Image:PCR diagram.png|1000px]]<br> | ||
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| '''Sample''' || '''Integrated Density''' || '''DNA μg/mL''' || '''Conclusion''' | | '''Sample''' || '''Integrated Density''' || '''DNA μg/mL''' || '''Conclusion''' | ||
|- | |- | ||
| PCR: Negative Control || 1658372 || | | PCR: Negative Control || 1658372 || 0.0 || Negative | ||
|- | |- | ||
| PCR: Positive Control || 3527419 || | | PCR: Positive Control || 3527419 || 2.0 || Positive | ||
|- | |- | ||
| PCR: Patient 1 ID 19185, rep 1 || 1173985 || | | PCR: Patient 1 ID 19185, rep 1 || 1173985 || 0.82 || Negative | ||
|- | |- | ||
| PCR: Patient 1 ID 19185, rep 2 || 1600503 || | | PCR: Patient 1 ID 19185, rep 2 || 1600503 || 1.02 || Negative | ||
|- | |- | ||
| PCR: Patient 1 ID 19185, rep 3 || 1621751 || | | PCR: Patient 1 ID 19185, rep 3 || 1621751 || 1.21 || Negative | ||
|- | |- | ||
| PCR: Patient 2 ID 88142, rep 1 || 927301 || | | PCR: Patient 2 ID 88142, rep 1 || 927301 || 0.20 || Negative | ||
|- | |- | ||
| PCR: Patient 2 ID 88142, rep 2 || | | PCR: Patient 2 ID 88142, rep 2 || 962092 || 0.23 || Negative | ||
|- | |- | ||
| PCR: Patient 2 ID 88142, rep 3 || | | PCR: Patient 2 ID 88142, rep 3 || 901293 || 0.19 || Negative | ||
|} | |} | ||
Latest revision as of 16:15, 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-UP
Initial Machine TestingThe images above depict our OpenPCR machine. It performs polymerase chain reactions (PCR), a process in which a particular DNA sequence is amplified. The DNA sequence can then be easily analyzed. The OpenPCR machine accomplishes the amplification of a DNA sequence through a series of heating and cooling sequences.
When we unplugged the LCD screen from the circuit board, the machine stopped displaying information on the LCD screen. When we unplugged the white wire that connects the circuit board to the heating block, the heating block would not heat up.
We first tested our OpenPCR machine (machine #6) on October 25, 2012. We ran the machine through a preprogrammed sample test run. At first we were not getting consistent temperature readings outputting to the LCD screen; however, after we rebooted the OpenPCR machine and ran the test again the machine worked perfectly.
Protocols
Polymerase Chain Reaction
SET UP:
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology
ResultsImageJ Software Processing
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