BME103:T930 Group 9

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(Protocols)
(Protocols)
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'''Polymerase Chain Reaction'''<br>
'''Polymerase Chain Reaction'''<br>
-
 
+
<br>
'''Steps to Run PCR'''<br>
'''Steps to Run PCR'''<br>
1.Connect the PCR machine to the computer.<br>
1.Connect the PCR machine to the computer.<br>
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7.Press start on the prgram to begin running the PCR.<br>
7.Press start on the prgram to begin running the PCR.<br>
8.Collect and record data at the completion of the trial.<br>
8.Collect and record data at the completion of the trial.<br>
-
 
+
<br>
 +
'''* Provided Mixing Components'''<br>
 +
a)TaqDNA polymerase (non-recombinant modified form) <br>
 +
b)MgCl2<br>
 +
c)dNTP's <br>
 +
d)reaction buffers (at optimal concentration for DNA template amplification)<br>
 +
<br>
'''Flourimeter Measurements'''<br>
'''Flourimeter Measurements'''<br>

Revision as of 13:11, 8 November 2012

BME 103 Fall 2012 Home
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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Image:BME494_Asu_logo.png

Contents

OUR TEAM

Name: Devraj Patel Open PCR Machine Engineer and Research & Development Scientist
Name: Devraj Patel
Open PCR Machine Engineer and Research & Development Scientist
Name: Andrew Hensley  Experimental Protocol Planner
Name: Andrew Hensley
Experimental Protocol Planner
Name: Nathalie Vitale  Experimental Protocol Planner
Name: Nathalie Vitale
Experimental Protocol Planner
Name: Ojeen Korkes  Research and Development Scientist
Name: Ojeen Korkes
Research and Development Scientist
Name: Brandon Simmons  Open PCR Machine Engineer
Name: Brandon Simmons
Open PCR Machine Engineer

LAB 1 WRITE-UP

(Please finish by 11/7/2012)

Initial Machine Testing

The Original Design
Image:PCRMachineImage.png
This is an image of an Open PCR Machine. This machine regulates the temperature of the DNA smaples both heating and cooling samples to the preset temperature and times set up beforehand using the set up program. This heating and cooling seperates the DNA strands at high heat and then allows it to synthesize at low temperature.


Experimenting With the Connections

When we unplugged part 3, the LCD, from part 6, the Open PCR circuit Board, the LCD on the machine turned off and no information appeared on the LCD screen.


When we unplugged the white wire that connects part 6, the Open PCR circuit Board, to part 2, the heat sink, the machine no longer accurately regulates and controls the temperature and result in malfunction of heating the PCR tubes.


Test Run

On October 25 2012, we conducted our first test on our open PCR machine. We tested the machine to test the operation functionality. The initial test demonstrated the machine heat sink accuratly controlled and displayed the preprogrammed temperaute determined by the software on the computer. The overall successfullness of the machine was good, however it came with one difficulity, fluctuation of time to complete the preporgrammed cycles.




Protocols

Polymerase Chain Reaction

Steps to Run PCR
1.Connect the PCR machine to the computer.
2.Open the 'OpenPCR' program on the computer.
3.Label the tubes. This information should include the patient number (1 or 2) or control (+ or -), as well as the replication number (1, 2, or 3).
4.Prepare the experiment by inserting the reactants into the PCR tubes. These tubes will consist of the patients DNA, along with the other provided mixing components*. After filling each tube, put it into the chamber at the top of the machine.
5.Close and tighten the lid of the chamber.
6.Customize the settings in the 'Thermal Cycler' program to include three stages: Stage 1 - one cycle that will heat the reactants up to 95 degrees Celcius for three minutes, Stage 2 - 35 cycles that will heat the reactancts to 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, and 72 degrees Celsius for 30 seconds.
7.Press start on the prgram to begin running the PCR.
8.Collect and record data at the completion of the trial.

* Provided Mixing Components
a)TaqDNA polymerase (non-recombinant modified form)
b)MgCl2
c)dNTP's
d)reaction buffers (at optimal concentration for DNA template amplification)

Flourimeter Measurements

(Add your work from Week 3, Part 2 here)




Research and Development

Specific Cancer Marker Detection - The Underlying Technology

In Week 3 of our PCR experiment, we studied the cancer marker in the PCR experiment was correlated to the breast and colorectal cancer. Based on conditional probabilities, we found that the frequency of this cancer found in Finland was 7.8%.

BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.

Summary: This is an image of how RNA primers bind to the cancer DNA template during the replication process. [1]
Summary: This is an image of how RNA primers bind to the cancer DNA template during the replication process. [1]
Source: [1]
Summary: This is an image of how Taq polymerases amplify the DNA. [2]
Summary: This is an image of how Taq polymerases amplify the DNA. [2]
Source: [2]


For more information on the PCR DNA replication process, please visit this website: http://learn.genetics.utah.edu/content/labs/pcr/.



Results

(Your group will add the results of your Fluorimeter measurements from Week 4 here)


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