BME103:T930 Group 10

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(Initial Machine Testing)
(Initial Machine Testing)
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'''The Original Design'''<br>
'''The Original Design'''<br>
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(Add image of the full OpenPCR machine here, from the Week 3 exercise. Write a paragraph description for visitors who have no idea what this is)<br>
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[[Image:BME103_Group10_Assembly.png‎|300px|3D CAD image of Open PCR Machine]]<br>

Revision as of 11:26, 4 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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Contents

OUR TEAM

Name: Nolan Bidese Role: Research and Development Specialist
Name: Nolan Bidese
Role: Research and Development Specialist
Name: Evan Austin Role: Open PCR Machine Engineer
Name: Evan Austin
Role: Open PCR Machine Engineer
Name: Aldin Malkoc Role: Open PCR Machine Engineer
Name: Aldin Malkoc
Role: Open PCR Machine Engineer
Name: Mikayle Holm Role: Experimental Protocol Planner
Name: Mikayle Holm
Role: Experimental Protocol Planner
Name: Coleen Fox Role: Experimental Protocol Planner
Name: Coleen Fox
Role: Experimental Protocol Planner

LAB 1 WRITE-UP

(Please finish by 11/7/2012)

Initial Machine Testing

The Original Design
3D CAD image of Open PCR Machine


Experimenting With the Connections

When we unplugged the circuit board from the mounting plate, the LED display on the PCR machine stopped functioning.

When we unplugged the white wire that connects the Open PCR circuit board to the heating plate, the temperature recordings that were displayed on the LED display stopped functioning.


Test Run

(Write the date you first tested Open PCR and your experience(s) with the machine)




Protocols

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a technique used to amplify fragments of DNA. This allows researchers to see the base sequence of the DNA. It works by the DNA polymerase enzyme synthesizes a complementary strand of the fragmented DNA when mixed with primers that signal where the DNA sequencing should begin. When the DNA polymerase enzyme, MgCL2, dNTP’s, forward primer, and reverse primers are all added to the test tubes and placed in the PCR machine, the mixture is first heated to separate the double helix, then cooled to allow the primers to bind. After the primers bind, the polymerase completes the new complementary strands. The PCR machine then repeats heating and cooling cycles to multiply the fragmented DNA. After a couple hours, the now amplified segments of DNA can be analyzed to test for a cancer marker.

Procedure:
1. Add a fragment of double stranded DNA, the pre-mixed Taq DNA polymerase, MgCl2, dNTPs, forward and reverse primers, and the DNA polymerase enzyme to test tube.
2. Place in PCR machine.
3. Set the following PCR cycle stages:
Stage 1: 1 cycle, 95 degrees Celsius for 180 seconds (separate the DNA double helix)
Stage 2: 30 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds (separate the DNA double helix, primers bind to single strands of DNA at 57 degrees Celsius, DNA polymerase enzyme adds bases to singles strands of DNA segments at 72 degrees Celsius, cycle is repeated so the DNA is multiplied)
Stage 3: 72 degrees for 180 seconds (final nucleotides are added)
Final Hold: 4 degrees Celsius
4. Run the PCR machine.
5. After about 2 hours, the cycles should be completed. DNA can now be used for research and testing for a cancer marker.

The PCR (GoTaq) Master Mix is advertised as a “ready-to-use solution” and it contains the Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers. These substances are mixed at proper concentrations so the user can achieve a useable amplification of DNA segments by PCR.

Reagent Volume
Template DNA (20 ng)0.1μL
10μM forward primer0.5μL
10μM reverse primer0.5μL
GoTaq master mix25.0μL
dH2O23.9μL
Total Volume50.0μL

Samples
Test tube 1-
Positive Control
Cancer DNA template
Test tube 2-
Patient 1
Replicate 1
ID: 30576
Gender: Male
Age: 46
Test tube 3-
Patient 1
Replicate 2
ID: 30576
Gender: Male
Age: 46
Test tube 4-
Patient 1
Replicate 3
ID: 30576
Gender: Male
Age: 46
Test tube 5-
Negative Control
DNA Template
Test tube 6-
Patient 2
Replicate 1
ID: 96210
Gender: Male
Age: 59
Test tube 7-
Patient 2
Replicate 2
ID: 96210
Gender: Male
Age: 59
Test tube 8-
Patient 2
Replicate 3
ID: 96210
Gender: Male
Age: 59

Flourimeter Measurements

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




Research and Development

Specific Cancer Marker Detection - The Underlying Technology

(Add a write-up of the information discussed in Week 3's class)

(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.)




Results

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


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