BME100 f2013:W900 Group1 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: Neema Jamali
Name: Neema Jamali
Name: Kaitlyn Allen
Name: Kaitlyn Allen
Name: Riddhi Rohit
Name: Riddhi Rohit
Name: Carter Hill
Name: Carter Hill
Name: Crawford Pederson
Name: Crawford Pederson

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

This is the Open PCR machine in its fully functional state. The machine uses a thermal cycler to have a small sample of DNA interact with added primers and Taq DNA polymerase in order to replicate desired strands of DNA at an exponential rate.


Experimenting With the Connections

When we unplugged (part 3) from (part 6), the display stopped working because it became disconnected from the power source. At the same time, the connection to the circuit that contains the code to make the display work also became disconnected. This cable supplies both the power to run the LCD display as well as transfers the information to be displayed on the screen. Without this connection the LCD display cannot operate.

When we unplugged the white wire that connects (part 6) to (part 2), the machine could not read the temperature of the micropipette holder. Thus, one would not be able to successfully perform PCR because of the obvious temperature discrepancies that would result. This white wire is actually a thermocouple which is used for measuring temperature. Without a correct temerature reading the OpenPCR is unable to regulate and change temperature to perform PCR.


Test Run

Started test run at 10:06 a.m. on October 23, 2013. The machine made it through 12 of the 35 cycles in an hour, with two hours and ten minutes remaining. The process appeared to run without any interruptions or complications, however the rate of progress was slower than expected.




Protocols

Thermal Cycler Program


DNA Sample Set-up

Positive Control (PC0) Patient ID 67618 Replicate 1 (PC1) Patient ID 67618 Replicate 2 (PC2) Patient ID 67618 Replicate 3 (PC3)
Negative Control (NC0) Patient ID 78379 Replicate 1 (NC1) Patient ID 78379 Replicate 2 (NC2) Patient ID 78379 Replicate 3 (NC3)


DNA Sample Set-up Procedure

  1. Step 1: Label the tubes.
  2. Step 2: Add PCR reaction and DNA/ primer mix to test tubes.
  3. Step 3: Run machine and collect data.


PCR Reaction Mix

PCR reaction, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's.


DNA/ primer mix

DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.




Research and Development

PCR - The Underlying Technology

Functions
Template DNA is just as it sounds, it is used as a template (guide) for the polymerase being used. It contains the target sequence. The function of the primers include attaching to sites on the DNA strands that are at either end of the segment that needs to be copied. The taq polymerase reads the DNA code and then attaches matching nucleoties to create DNA copies. Magnesium chloride is used as a catalyst during the reaction to help speed up the process. Deoxyribonucleotides are the building blocks of the new strand, that is what the strand is made out of.

Steps of Thermal Cycling
The first step of thermal cycling occurs at a temperature of 95 degrees C and sets the stage for the entire process. At this initial step, which takes 3 minutes to complete, the DNA polymerase activates by heating up and prepares to unwind. During the denature step at 95 degrees C for 30 seconds, the DNA double helix seperates creating two single-stranded DNA molecules. Next, during the anneal stage at 57 degrees C for 30 seconds, a single stranded DNA molecule naturally attempts to pair up. Furthermore, at the extend stage at 72 degrees C for 30 seconds the DNA polymerase is activated and adds complementary nucleotides onto the strand. Lastly. during the final stage at 72 degrees C for 3 minutes there are fragments left that only contain the target sequence of DNA. The final hold is an additional step in thermal cycling and takes place at 4 degrees C. This step is used to make sure that the DNA does not unwind or degenerate after all other steps have been done.

DNA Pairing
Adenine, which is a purine, pairs with thymine, a pyrimidine. Guanine, which like adenine is also a purine, pairs with cytosine, a pyrimidine like thymine.


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