BME103:T930 Group 2: Difference between revisions
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|style="background-color: #FFF0F5" | [[BME103 | <font face="trebuchet ms" style="color: #000000"> '''Home''' </font>]]<br>[[BME103:People | <font face="trebuchet ms" style="color: #000000"> '''People''' </font>]]<br>[[BME103:Projects | <font face="trebuchet ms" style="color: #000000"> '''Lab Write-Up 1''' </font>]]<br>[[BME103:Projects2 | <font face="trebuchet ms" style="color: #000000"> '''Lab Write-Up 2''' </font>]]<br>[[BME103:Projects3 | <font face="trebuchet ms" style="color: #000000"> '''Lab Write-Up 3''' </font>]]<br>[[BME103:Logistics | <font face="trebuchet ms" style="color: #000000"> ''' Course Logistics For Instructors''' </font>]] <br>[[BME103:Photos | <font face="trebuchet ms" style="color: #000000"> '''Photos''' </font>]] <br>[[BME103:WikiHelp | <font face="trebuchet ms" style="color: #000000"> '''Wiki Editing Help''' </font>]] | |style="background-color: #FFF0F5" | [[BME103 | <font face="trebuchet ms" style="color: #000000"> '''Home''' </font>]]<br>[[BME103:People | <font face="trebuchet ms" style="color: #000000"> '''People''' </font>]]<br>[[BME103:Projects | <font face="trebuchet ms" style="color: #000000"> '''Lab Write-Up 1''' </font>]]<br>[[BME103:Projects2 | <font face="trebuchet ms" style="color: #000000"> '''Lab Write-Up 2''' </font>]]<br>[[BME103:Projects3 | <font face="trebuchet ms" style="color: #000000"> '''Lab Write-Up 3''' </font>]]<br>[[BME103:Logistics | <font face="trebuchet ms" style="color: #000000"> ''' Course Logistics For Instructors''' </font>]] <br>[[BME103:Photos | <font face="trebuchet ms" style="color: #000000"> '''Photos''' </font>]] <br>[[BME103:WikiHelp | <font face="trebuchet ms" style="color: #000000"> '''Wiki Editing Help''' </font>]] | ||
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| style="background-color: # | | style="background-color: #000000; padding: 5px;" colspan="2" | [[Image:ASUlogo3.png]] | ||
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|- valign="top" | |- valign="top" | ||
| [[Image:BME103 Group2 Ryan S.JPG |130px|thumb|Ryan Sullivan<br>Research Development Scientist]] | | [[Image:BME103 Group2 Ryan S.JPG |130px|thumb|Ryan Sullivan<br>Research Development Scientist]] | ||
| [[Image: | | [[Image:Miriam_acosta_103_group22.jpg.jpg|150px|thumb|Miriam Acosta<br>PCR Machine Engineer]] | ||
| [[Image:BME103_Group2_RyanKeeney.JPG|130px|thumb|Ryan Keeney<br>PCR Machine Engineer]] | | [[Image:BME103_Group2_RyanKeeney.JPG|130px|thumb|Ryan Keeney<br>PCR Machine Engineer]] | ||
| [[Image:BME103Julie.jpg|150px|thumb|Juliana Ramos<br>Experimental Protocol Planner]] | | [[Image:BME103Julie.jpg|150px|thumb|Juliana Ramos<br>Experimental Protocol Planner]] | ||
| [[Image: | | [[Image:Aarons small.jpg|130px|thumb|x120px|Aaron Cornejo<br>Experimental Protocol Planner]] | ||
|} | |} | ||
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==Initial Machine Testing== | ==Initial Machine Testing== | ||
[[Image:BME103_Group2_OpenPCRScreenshotlabels.jpg|thumb|upright=2|alt=Open PCR.|Open PCR.]] | [[Image:BME103_Group2_OpenPCRScreenshotlabels.jpg|thumb|upright=2.5|alt=Open PCR.|Open PCR.]] | ||
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'''Polymerase Chain Reaction'''<br> | '''Polymerase Chain Reaction'''<br> | ||
A PCR (polymerase chain reaction), according to [http://www.dnalc.org/resources/animations/pcr.html Cold Spring Harbor Laboratory], “enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours. This automated process bypasses the need to use bacteria for amplifying DNA.” Thus forth, it essentially does go through a biochemical process to replicate the desired sequence of DNA. The PCR machine has the capacity to isolate and amplify that desired strand of DNA through the repeated cycling of cooling and heating. | |||
'''Thermal Cycler Program:'''<br> | |||
Stage one: 1 cycle, 95°C for 3 minutes | |||
Stage two: 35 cycles, 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds | |||
Stage three: 72°C for 3 minutes | |||
Final hold: 4°C | |||
{| border="1" style="border-collapse:collapse;" style="margin: 1em auto 1em auto;" | |||
|- | |||
! Reagant !! Volume | |||
|- | |||
| Template DNA (20 ng) || 0.2 μL | |||
|- | |||
| 10 μM Forward Primer || 1.0 μL | |||
|- | |||
| 10 μM Reverse Primer || 1.0 μL | |||
|- | |||
| GoTaq master Mix || 50.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 47.8 μL | |||
|- | |||
! Total Volume || 100.0 μL | |||
|} | |||
<div style="text-align: center;"> Patient 1 </div> | |||
{| border="1" style="border-collapse:collapse;" style="margin: 1em auto 1em auto;" | |||
|- | |||
! Sample !! ID !! Sex !! Age !! Date/Time | |||
|- | |||
| Control || 15308 || M || 61 || Thursday 9:30 AM | |||
|- | |||
| 1-1 || 15308 || M || 61 || Thursday 9:30 AM | |||
|- | |||
| 1-2 || 15308 || M || 61 || Thursday 9:30 AM | |||
|- | |||
| 1-3 || 15308 || M || 61 || Thursday 9:30 AM | |||
|} | |||
<div style="text-align: center;"> Patient 2 </div> | |||
{| border="1" style="border-collapse:collapse;" style="margin: 1em auto 1em auto;" | |||
|- | |||
! Sample !! ID !! Sex !! Age !! Date/Time | |||
|- | |||
| Control || 36372 || F || 56 || Thursday 9:30 AM | |||
|- | |||
| 2-1 || 36372 || F || 56 || Thursday 9:30 AM | |||
|- | |||
| 2-2 || 36372 || F || 56 || Thursday 9:30 AM | |||
|- | |||
| 2-3 || 36372 || F || 56 || Thursday 9:30 AM | |||
|} | |||
<BR> | |||
'''Flourimeter Measurements'''<br> | '''Flourimeter Measurements'''<br> | ||
[[Image:BME103 WaterDropplet.jpg|thumb|upright=1|alt=Open PCR.|Droplet of water on hydrophobic slide.]] | [[Image:BME103 WaterDropplet.jpg|thumb|upright=1|alt=Open PCR.|Droplet of water on hydrophobic slide.]] | ||
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3. Open the front flap of the black box.<br> | 3. Open the front flap of the black box.<br> | ||
4. Place hydrophobic/glass slide<br> | 4. Place hydrophobic/glass slide<br> | ||
5. Using pipette to obtain droplets of SYBR green dye | 5. Using pipette to obtain droplets of SYBR green dye<br> | ||
6. Proceed to squeeze two drops of dye on the slide.<br> | 6. Proceed to squeeze two drops of dye on the slide.<br> | ||
7. Obtain DNA sample. <br> | 7. Obtain DNA sample. <br> | ||
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[[Image:Dna_primerGroup2.png|600px|]] | [[Image:Dna_primerGroup2.png|600px|]] | ||
<BR> [http://dr282zn36sxxg.cloudfront.net/datastreams/f-d%3Afae9420fc09f07a55c408edd6b1bd792762100d04e09b00b9d1ea612%2BIMAGE%2BIMAGE.1 *Imagine from above with labels] | |||
With the r17879961 DNA that was sequenced, the primer that was made for the DNA contained a strand of nucleotides that went: "ATGTGACGT". | |||
[[Image:Primer22.jpg|600px|]] | |||
These matched up with the section of the DNA in the 22nd chromosome if the sequence did indicate cancer. The cancer sequence would be complimentary to the primer, it is: "TACACTGCA". This would yield a positive result | |||
[[Image:Cancercode.jpg|600px|]] | |||
A non-cancerous sequence is shown below ("TACATTGCA"), which would not attach to the primer and would yield a negative result. | |||
[[Image:Ideal.jpg|600px|]] | |||
When the primer attaches to the cancerous sequence, it works, and the replication process can begin: | |||
[[Image:Cancer+primer.jpg|600px|]] | |||
When the primer tries to attach to a non-cancerous sequence, it fails, because the "T" and "G" do NOT connect, and therefore the replication process is not exponential: | |||
[[Image:Ideal+primer.jpg|600px|]] | |||
<br><br> | <br><br> | ||
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==Results== | ==Results== | ||
[[Image:BME103_Group2_CALFTHYMUS.jpg|150px| | |||
[[Image:BME103_Group2_WATER.jpg|150px| | |||
[[Image:BME103_Group2_SAMPLE_1-1.jpg|150px|Patient ID 15308 Sample 1]] | |||
[[Image:BME103_Group2_SAMPLE_1-2.jpg|150px|Patient ID 15308 Sample 2]] | [[Image:BME103_Group2_CALFTHYMUS.jpg|150px||thumb|upright| Positive Control]] | ||
[[Image:BME103_Group2_SAMPLE_1-3.jpg|150px|Patient ID 15308 Sample 3]] <br><br> | [[Image:BME103_Group2_WATER.jpg|150px|thumb|upright|Negative Control]] <br><br> | ||
[[Image:BME103_Group2_SAMPLE_2-1.jpg|150px|Patient ID 36372 Sample 1]] | |||
[[Image:BME103_Group2_SAMPLE_2-2.jpg|150px|Patient ID 36372 Sample 2]] | |||
[[Image:BME103_Group2_SAMPLE_2-3.jpg|150px|Patient ID 36372 Sample 3]] | [[Image:BME103_Group2_SAMPLE_1-1.jpg|150px|thumb|upright|Patient ID 15308 Sample 1]] | ||
[[Image:BME103_Group2_SAMPLE_1-2.jpg|150px|thumb|upright|Patient ID 15308 Sample 2]] | |||
[[Image:BME103_Group2_SAMPLE_1-3.jpg|150px|thumb|upright|Patient ID 15308 Sample 3]] <br><br> | |||
[[Image:BME103_Group2_SAMPLE_2-1.jpg|150px|thumb|upright|Patient ID 36372 Sample 1]] | |||
[[Image:BME103_Group2_SAMPLE_2-2.jpg|150px|thumb|upright|Patient ID 36372 Sample 2]] | |||
[[Image:BME103_Group2_SAMPLE_2-3.jpg|150px|thumb|upright|Patient ID 36372 Sample 3]] | |||
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* '''Conclusion''' = Whether exponential DNA replication has occured | * '''Conclusion''' = Whether exponential DNA replication has occured | ||
Given the results of all the DNA testing and evaluation we conclude that Patient 1 (ID 15308) contains the cancerous gene tested for, and that Patient 2 (ID 36372) did not. | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
Latest revision as of 14:40, 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 AWESOME TEAMLAB 1 WRITE-UPInitial Machine Testing
Unplugging the LED interface cable from the processing chip caused the display of the machine to turn off. The blank screen meant that the machine stopped sending data to display. Unplugging anything from the circuit board causes it not to work because no electricity can be sent.
Test Run Our initial testing of the PCR machine went quite well. We used the Open PCR software to program our custom cycle quite easily. Once the PCR was connected to the computer via USB we continued to run the trial. During the run we made sure that the temperature displayed on the LED display match the temperature on the computer program. The temperatures matched the entire duration of the experiment. The test run carried out flawlessly, just a bit slowly.
ProtocolsPolymerase Chain Reaction
Stage one: 1 cycle, 95°C for 3 minutes Stage two: 35 cycles, 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds Stage three: 72°C for 3 minutes Final hold: 4°C
Patient 1
Patient 2
Flourimeter Assembly Instructions: 1. Gather materials (PCR Machine, tools optional)
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology The r17879961 DNA was sequenced to determine the chain of nucleotides that it is composed of. A section of this DNA shows a change in the allele sequence; one which might indicate cancer. In the sequence, a change from "ATT" to "ACT" is the specific indication marker for cancer. The artificial primer that is introduced to the DNA chain is meant to be a complimentary strand that matches up with the "ACT" and surrounding nucleotide sequence ("TGA"). When the primer attaches successfully, it allows the Taq polymerase to finish attaching nucleotides to the sequence. The number of strands will be doubled in this case, and by using the PCR replication machine, the sample will be heated and cooled in specific intervals. These intervals are meant to manipulate the DNA and do things such as separate a strand, cool it down so it is able to bind, as well as stretch it out. This process will, ultimately, result in exponential DNA replication. If the primer does not attach successfully, then this means the person does not have a cancer-indicating sequence, and it will, therefore, not replicate the DNA exponentially. If the DNA replicates exponentially, then the fluorescent dye will show, and this would indicate a cancerous genome.
The illustration below shows the PCR process when the primer attaches to the DNA and the Taq polymerase and is able to complete the DNA chain, in coordination with the PCR machine temperature cycles. It is shown that throughout the process, the DNA replicates at an exponential rate.
These matched up with the section of the DNA in the 22nd chromosome if the sequence did indicate cancer. The cancer sequence would be complimentary to the primer, it is: "TACACTGCA". This would yield a positive result A non-cancerous sequence is shown below ("TACATTGCA"), which would not attach to the primer and would yield a negative result. When the primer attaches to the cancerous sequence, it works, and the replication process can begin: When the primer tries to attach to a non-cancerous sequence, it fails, because the "T" and "G" do NOT connect, and therefore the replication process is not exponential:
Results
Given the results of all the DNA testing and evaluation we conclude that Patient 1 (ID 15308) contains the cancerous gene tested for, and that Patient 2 (ID 36372) did not.
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