BME100 f2013:W900 Group15 L4

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BME 100 Fall 2013 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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Contents

OUR TEAM

Name: Justin DombrowskiProtocol Planning
Name: Justin Dombrowski
Protocol Planning
Name: Saiswathi JavangulaResearcher and Developer
Name: Saiswathi Javangula
Researcher and Developer
Name: Gage BebakOpen PCR Machine Testing
Name: Gage Bebak
Open PCR Machine Testing
Name: Ryan FisherProtocol Planning
Name: Ryan Fisher
Protocol Planning
Name: AbdulrahmanResearcher and Developer
Name: Abdulrahman
Researcher and Developer
Name: studentRole(s)
Name: student
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design


Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine did not shut off but all power to the LCD screen was cut off.

When we unplugged the white wire that connects (part 6) to (part 2), the machine could no longer sense the temperature of the heating and cooling plates properly because it was giving a -40.0 degrees celsius which is impossible because the machine was not activated nor was the surrounding room temperature below freezing.


Test Run

October 23, 2013: the initial test presented many problems with the machine. although it gave the correct time to complete the 35 cycles, the heating system never seemed to start working because it stayed on the same cycle the entire two hours and the temperature never increased or decreased thus proving that the machine and open PCR were not functioning properly.




Protocols

Thermal Cycler Program
1. The program starts with the initial step of a single cycle at 95°C for three minutes.

2. The next phase consists of 35 cycles; 95°C for 30 seconds to further the process of denaturation, then there is the anneal step which involves the temperature being 57°C for 30 seconds, and then the extended step at 72°C for 30 seconds.

3. The final step is set at 72°C for 3 minutes.

4. A refrigeration step of the DNA takes place during the final hold at 4°C.

DNA Sample Set-up

PC
Positive Control- cancer DNA template
P11
Patient 1, id-78086, Replicate 1
P12
Patient 1, id-78086, Replicate 2
P13
Patient 3, id-78086, Replicate 1
NC
Positive Control- non-cancer DNA template
P21
Patient 2, id-32445, Replicate 1
P22
Patient 2, id-32445, Replicate 2
P23
Patient 2, id-32445, Replicate 3


DNA Sample Set-up Procedure
1. Label each tube in accordance with the above table to identify the controls, patients, and replicates.
2. Insert the DNA/primer mixes (15μL) into its respective tubes and add the PCR reaction mixes (15μL) to each of the tubes.
3. Run the OpenPCR machine and collect data.


PCR Reaction Mix

  • Taq DNA polymerase, MgCL2, and dNTP's


DNA/ primer mix

  • Each contains different template DNA's but all have the same forward primer and reverse primer.





Research and Development

PCR - The Underlying Technology

Function of each component in PCR reaction"'

There are many components to PCR reaction. The function of the template DNA during PCR reaction is to create a complimentary strand to result in one DNA strand that will convert into two. Primers are used when a strand of DNA attaches to a segment of interest that needs to be copied while the primers also isolate the DNA part of interest. attaches to the bottom strand of the other end. The Taq polymerase copies a cell's DNA before it divides it into two. Magnesium chloride is important to the reaction as it is added to allow the presence of magnesium into the reaction as it acts a catalyst as the reaction cannot proceed without magnesium. The deoxyribonucleotides grab nucleotides that are floating in the liquid around it and attaches them to the end of the primer.

Steps of thermal cycling

During the initial step of 95 degrees Celsius for 3 minutes, the DNA separates from its double helix formation, allowing the two strands of DNA to be parallel to one another. During the step of Denature at 95 degrees Celsius for 30 seconds, the double stranded DNA separates or denatures into single strands. Next, is the process of anneal at 57 degrees Celsius for 30 seconds, the primers bind or anneal to complimentary matches on the target DNA sequence. During the next step of extend, the enzyme Taq polymerase binds to the prime sequences and adds nucleotides to extend the second strand. During the final step at 72 degrees celsius for 3 minutes, the target sequence defined by the primers begins to accumulate. During the final hold of four degrees Celsius, the remaining DNA left in the sample after the cycling is identified. During the base-pairing of the bonding, Adenine (A) sticks to Thymine (T), while Cytosine (C) sticks to Guanine (G).

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Source: http://www.ucl.ac.uk/~ucbhjow/b200/pcr.htm


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