BME100 f2013:W1200 Group8 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: Colby MarkResearch and Development Specialist
Name: Colby Mark
Research and Development Specialist
Name: Hayden McIverOpen PCR Machine Testing
Name: Hayden McIver
Open PCR Machine Testing
Name: Isai ValdezProtocol Planning
Name: Isai Valdez
Protocol Planning
Name: Madison GraysonProtocol Planning
Name: Madison Grayson
Protocol Planning
Name: Andrew WillsOpen PCR Machine Testing
Name: Andrew Wills
Open PCR Machine Testing

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:Group8-PM-PCR.JPG‎Image:PCR5.jpg

OpenPCR is a themocycler used to conduct heat through samples of data. This is used to replicate DNA through a series of heating and cooling in order to allow certain chemical reactions to occur.The design is made to prevent any outside reagents from affecting the concentrations of the samples; certain features such as precise temperature control and a heated lid allow there to be no interference with the data. The OpenPCR conveniently connects to your computer and stores the data from every trial.


Experimenting With the Connections

When we unplugged the LCD screen (part 3) from the circuit board (part 6), the machine's LCD screen turned off because power was cut off.

When we unplugged the white wire that connects the circuit board (part 6) to heating block (part 2), the machine showed a drop in temperature because the heating element lost power.

Test Run
The date that we first tested the Open PCR was on October 23rd, 2013. We didn't think that we would be able to open up the panel and see the inner workings of this machine. We thought it was very interesting to see how the wires are connected and also how the cooling and heating system works with the layered Aluminum plates. The process of completing the entire lab did take quite a long time, but compared to other machines ours worked to perfection.




Protocols

Thermal Cycler Program
Heated Lid: 100 degrees Celsius
Initial Step: 95 degrees Celsius for 3 mins
Step: 35 cycles
-Denature: 95 degrees C for 30 sec
-Anneal: 57 degrees C for 30 sec
-Extend: 72 degrees C for 30 sec
Final Step: 72 degrees C for 3 mins
Final Hold: 4 degrees C

DNA Sample Set-up

Cancer DNA template (C+) patient 1 ID:21489 Tube: 1R1 Patient 1 ID: 24189 Tube: 1R2 Patient 1 ID: 24189 Tube: 1R3
Non-cancer DNA template (C-) Patient 2 ID:90434 Tube: 2R1 Patient 2 ID:90434 Tube: 2R2 Patient 2 ID:90434 Tube: 2R3


DNA Sample Set-up Procedure

  1. Step 1. Label each tube to avoid using wrong substance.
  2. Step 2. Dispense 50 ul of PCR reaction mix using pipettes into each of the 8 tubes
  3. Step 3. Dispense 50 ul of each DNA sample in their respective tubes, switching to a new tip when a different substance is handled to prevent cross contamination.
  4. Step 4. Place tubes in PCR machine and close the lid.
  5. Step 5. make sure PCR machine is programmed to the right temperatures and cycles.
  6. Step 6. Connect the PCR machine to the computer.
  7. Step 7. Run the Thermo cycler program on the Open PCR software.


PCR Reaction Mix

  • The PCR reaction mix contains the following:

-Taw DNA polymerase -MgCl2 -dNTP


DNA/ primer mix

  • -A forward primer

-A reverse Primer





Research and Development

PCR - The Underlying Technology


Functions of each component of a PCR Reaction
The first step is to gather Template DNA this is a double stranded DNA that is separated and then used to form the replicated DNA. Then primers are added to the solution and these are man-made DNA sequences that contain nucleotides which bind to part of the DNA segment that we want to be replicate. The next step is to add TAQ Polymerase these are sequences of proteins similar to an enzyme that attaches to the primer and adds the nucleotides to the segment where the primers are located in order to replicate the DNA. There are also ways to speed and slow down the replication process, by adding Magnesium Chloride. Magnesium acts as a catalyst to speed up the reaction by binding the taq polymerase to the primers and DNA. There should not be too little as this will cause the binding to slow, or stop all together. If there is too much, the DNA replication process with begin working too fast and could skip sequences, which would lead to errors in replicating the strands. Lastly, Deoxyribonucleotides which are DNA nucleotides base pair with the original DNA strand with the help of the TAQ Polymerase to form copy DNA.

Each step of thermal Cycling
There is the initial step which occurs at 95 degrees Celsius for three minutes. During this step, the double helix DNA structure unwinds. The Denature step which occurs next is at 95 degrees Celsius for 30 seconds. During this step, the template DNA separates into single stranded DNA. After denaturing, the Anneal process starts. This occurs at 57 degrees Celsius for another 30 seconds. During this process, the Taq Polymerase binds and begins to copy the DNA by adding nucleotides. The Final step, which is at 72 degrees Celsius for three minutes, is when the DNA extension or "copying" is completed. The very last process that may or may not be needed is the Final Hold at 4 degrees Celsius. During this step it stops the activity of taq polymerase and prevents unwanted copying.

Nucleotide Base Pairing
DNA is made up of four types of molecules called nucleotides. They are designated as A,T,C, and G. The pairing of these bases is driven by hydrogen bonding, and this allows the DNA strands to stick together. The bases are Adenine (A), which sticks to Thymine (T). And Cytosine (C), which sticks to Guanine (G).






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