BME103 s2013:T900 Group8: Difference between revisions
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| [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]] | | [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]] | ||
| [[Image: | | [[Image:456246 3499926058852 287604656 o.jpg|thumb|Name: Josh Snyder<br>Role: Machine Tester]] | ||
| [[Image: | | [[Image:BME103 Group8 PAK.jpg.jpg|thumb|Name: Adam Pak<br>Role: Experimental Protocol Planner]] | ||
| [[Image:BME103_Group8_Sunshine.jpg|thumb|Name: Sunshine Silvas<br>Role: Machine Testing]] | | [[Image:BME103_Group8_Sunshine.jpg|thumb|Name: Sunshine Silvas<br>Role: Machine Testing]] | ||
| [[Image: | | [[Image:Ew.png]]Name: Renee Tran<br>Role: Research and Development Scientist | ||
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==Initial Machine Testing== | ==Initial Machine Testing== | ||
[[Image:BME103_Group8 PCR.JPG|150px|thumb|Open PCR Machine used in Lab]]<br> | |||
'''The Original Design'''<br> | '''The Original Design'''<br> | ||
The PCR Machine otherwise known as a Thermocycler or a DNA Amplifyer is use by scientists to create a vast quanity of a specific sequence of DNA. This method is also used widely for biological and medical applicaions. This process relies on the thermal cycles to amplify the DNA to then be able to replicated it. The system is run by means of a software downloaded to either a desktop or laptop. By connecting the PCR machine to a computer it is then possible to run a test with the unique specifications needed; such as tempurature and number of cycles.<br><br>[[Image:PCR_group3_.png|10px|thumb|frame|left|PCR diagram]] '''Experimenting With The Connections'''<br> | |||
When taking apart the PCR Machine there are pieces that if not connected properly or were not working apropriatly would cause the test to be inacurate and result in unreliable data.<br> By umpluging the LCD screen from everything this else truned off the screen and we were unable to see the process that the machine was running. Another part of the machine that was important to make sure it was working properly was the battery, motherboard, and tempurature sensor. When the tempurature sensor was unpluged the reading on the LED screen was inaccurate. Once it was reconnected the tempurature retured to the apropriate level and the reading on the screen was reliable. Each of these components are importante and if they were not working properly could cause the test runs to come out negative and false.<br><br>'''Test Run''' | |||
The first test date was March 5. It began around 10:00am and completed the 4 minute test cycle with no issues.<br><br> | |||
A preliminary test on March 19 concerned the Machine testers as the PCR Machine was not completing the specifies cycle. The machine was also remaining above 100 degrees and would not cool down to meet the requirements of the test.<br><br> | |||
After more testing, it was clear to the group that machine 1 was not going to produce reliable results based on the variation in temperature and the inability of the machine to complete the cycle. The group then moved the test samples to the PCR Machine that another group was testing so that the test might be run on a reliable machine. The machine tester of this new group must be relied upon to make sure that the new machine is operational and will produce reliable results.<br> | |||
<br><br> | <br><br> | ||
==Protocols== | ==Protocols== | ||
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6. Step 6 : Start the PCR machine using software | 6. Step 6 : Start the PCR machine using software | ||
'''PCR Reaction Mix''' | '''PCR Reaction Mix''' and | ||
'''DNA/ primer mix''' <br> | |||
Inside the PCR reaction mix there are six different components. The first is the template, the template is the specific type of DNA or RNA the person wants to replicate. The quality of the template can determine the outcome of the PCR machine. The seconded is the primers, a person can choice many different types of primers, but they all accomplish the same goal they prime the DNA to be ready for replication. The Third piece of the mix is a DNA polymerase; the polymerase greatly affects the outcome of the PCR machine because this is the enzyme that replicates the DNA or RNA. Fourth is the MgCl2 concentration, this concentration is key it soluble complexes with dNTPs to produce the sub straight that the polymerase will recognize. The fifth is Deoxynucleotide triphosphate (dNTP) concentration and lastly is the pH of the solution. The pH is important because there is a sweet spot that produces the maximum results; this is around a pH of 8.3-9.0. Inside the DNA/ primer mix are a few things. The first in the DNA mix is the template strand of DNA that is going to be replicated. Also there is the DNA polymerase that once active will construct the new DNA strand. The primer mix is an enzyme that attaches to the first three nucleotides in a DNA base and thus letting the polymerase attach more nucleotides to the free 3’ end. <br><br> | |||
'''DNA/ primer mix''' | sources : | ||
http://www.roche-applied-science.com/sis/amplification/pcr_amplification_050300.html http://www.contexo.info/DNA_Basics/Primers.htm | |||
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'''Specific Cancer Marker Detection - The Underlying Technology'''<br> | '''Specific Cancer Marker Detection - The Underlying Technology'''<br> | ||
The goal of this experiment is to understand the DNA amplification process in order to detect cancerous genes when given Template DNA; otherwise known as DNA taken from the patient. DNA amplification will allow the cancerous genes to replicate. The cancerous gene will produce a positive result, while the non-cancer gene will give a negative result, because the primers are designed to amplify cancerous DNA. Therefore, the cancerous mutation cannot bind to normal DNA, ultimately meaning that amplification cannot occur. | The goal of this experiment is to understand the DNA amplification process in order to detect cancerous genes when given Template DNA; otherwise known as DNA taken from the patient. DNA amplification will allow the cancerous genes to replicate. The cancerous gene will produce a positive result, while the non-cancer gene will give a negative result, because the primers are designed to amplify cancerous DNA. Therefore, the cancerous mutation cannot bind to normal DNA, ultimately meaning that amplification cannot occur.<br> | ||
<br> | <br> | ||
'''Functions of each component of the PCR Chain Reaction:'''<br> | |||
''Template DNA:'' The DNA taken from the patient<br> | |||
''Primers:'' Short (20 base pairs) pieces of synthesized DNA<br> | |||
''Taq Polymerase:'' is an enzyme that detects the primer-template DNA and snaps the dNtp's back to DNA<br> | |||
''Magnesium Chloride (MgCl2):'' serves as a "co-factor" for Taq Polymerase<br> | |||
''dNTP's:'' Once the DNA is "unzipped"and recombined the dNTP's are the unincorporated bases<br> | |||
''The following “3 steps” are repeated for 30 cycles, each step taking about 30 seconds.''<br> | ''The following “3 steps” are repeated for 30 cycles, each step taking about 30 seconds.''<br> | ||
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Ultimately, the cancerous DNA undergoes an exponential growth illustrated by the following:<br> | |||
[[Image:Exponential.png|700px]]<br> | |||
Source: http://amrita.vlab.co.in/?sub=3&brch=186&sim=321&cnt=1 | |||
Latest revision as of 21:30, 31 March 2013
BME 103 Spring 2013 | Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OUR TEAM
LAB 1 WRITE-UPInitial Machine TestingThe Original Design
Experimenting With The Connections When taking apart the PCR Machine there are pieces that if not connected properly or were not working apropriatly would cause the test to be inacurate and result in unreliable data. The first test date was March 5. It began around 10:00am and completed the 4 minute test cycle with no issues.
Protocols
Our job as for Experiment Protocol Planner was to :
We have educated our self via “PCR Virtual Lab” that can be found at http://openpcr.ord/use-it/ then downloaded and test software that was responsible for taking the results from PCR machine and displaying those results on PC, as well as to make commends to the PCR machine. This software also gave us real time update of the PCR machine PCR experiment summery
"Contains" describes what sample was inserted in the test tube DNA Sample Set-up Procedure 1. Step 1 : Acquire Materials
2. Step 2 : Mix dyes
3. Step 3 : Prepare the samples, and label the samples (tables is shown above)
6. Step 6 : Start the PCR machine using software PCR Reaction Mix and
DNA/ primer mix
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology The goal of this experiment is to understand the DNA amplification process in order to detect cancerous genes when given Template DNA; otherwise known as DNA taken from the patient. DNA amplification will allow the cancerous genes to replicate. The cancerous gene will produce a positive result, while the non-cancer gene will give a negative result, because the primers are designed to amplify cancerous DNA. Therefore, the cancerous mutation cannot bind to normal DNA, ultimately meaning that amplification cannot occur. The following “3 steps” are repeated for 30 cycles, each step taking about 30 seconds. • What happens? • What happens? • What happens?
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