BME100 f2013:W900 Group2 L4

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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: Leo SantosRole: Protocol Planner
Name: Leo Santos
Role: Protocol Planner
Name: Steven NyeRole: Researcher and Developer
Name: Steven Nye
Role: Researcher and Developer
Name: Robert RyeRole: Open PCR Machine Tester
Name: Robert Rye
Role: Open PCR Machine Tester
Name: John RichardsRole: Open PCR Machine Tester
Name: John Richards
Role: Open PCR Machine Tester
Name: Vivian VuongRole: Researcher and Developer
Name: Vivian Vuong
Role: Researcher and Developer

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

Image:300px-Labeled_OpenPCR.png‎
Image:openpcr1.png
  • Image from www.forbes.com


The OpenPCR machine is a very cost-efficient method of creating nearly-pure DNA samples that will have the DNA of any given sequence. In Laman's terms, this is a machine that can target a DNA sequence, replicate it, and then show if there was a reaction. This method would be used to find target sequences of DNA in any given sample, which can include precursor genetic sequences to cancer or even genetic diseases. Because of this, the OpenPCR machine can be used to detect such diseases from an early stage in a cost-efficient manner.

Experimenting With the Connections

When part 3 was unplugged from part 6, the machine's screen turned off. This would imply that this is the power source for the screen readout.

The white wire connecting part 6 to part 2 was the temperature sensor, so when unplugged, the machine became unable to read temperature, and the temperature changed from 0 degrees Celsius to -63.9 degrees Celsius.


Test Run

The first test of the OpenPCR machine on October 23, 2013 at 10:07 AM. The machine was in working order and performed to the expectations given by our instructors. The machine performed 22 cycles of DNA replication in one hour and seven minutes. There was no malfunction in heat cycles or starting the machine.



Protocols

Thermal Cycler Program
In order for PCR to take place, we need to run a program that will produce a certain heating/cooling protocol.
Step 1: 95°C for 3 minutes.
Step 2: 35 cycles, 95°C for 30 seconds, 57°C for 30 second, and 72°C for 30 seconds.
Step 3: 72°C for 3 minutes.
Final Hold: 4°C.

DNA Sample Set-up
Image:Dnasample.png


DNA Sample Set-up Procedure

  1. Gather all required materials.
  2. Add the DNA/primer mix to one of the PCR reaction mix.
  3. Repeat Step 2 for all of the DNA/primer and PCR reaction tubes.
  4. Make sure to use different pipette tips each time to not cross-contaminate the samples.
  5. Place the PCR tubes into the machine.
  6. Clean up all materials.


PCR Reaction Mix

  • There are 8 PCR reaction mix tubes. Each contains taq DNA polymerase, MgCl2, and dNTP's.

DNA/ primer mix

  • There will be 8 DNA/ primer mix tubes. Each contains a different template DNA. All tubes have the same forward primer and reverse primer.




Research and Development

PCR - The Underlying Technology


Components Of a PCR Reaction

Components of a PCR reaction include a template DNA, primers, taq polymerase, magnesium chloride and deoxyribonucleotides. Template DNA is the "master copy" used to replicate and make copies of DNA. Primers are created to match any kind of specific nucleotide sequence a complementary DNA strand needs. They attach to either end of a DNA segment they want to copy. Taq polymerase binds to the DNA strand and primer. It then reads DNA code and attaches matching nucleotides to create DNA copies. These nucleotides called deoxyribonucleotides are the building blocks of the DNA molecules.They are made up of a deoxyribose sugar, a base and a phosphate group. Magnesium chloride acts as a cofactor of taq polymerase during the PCR process.


PCR Thermal Cycling Process

The process of PCR includes 6 different phases: the initial step (95° C for 3 minutes), denature (95° C for 30 seconds), anneal (57° C for 30 seconds), extend (72° C for 30 seconds), the final step (72° C for 3 minutes), and the final hold (4° C). The initial step consists of the machine heating up for the PCR process to begin and the helix of the DNA unwinding. After 3 minutes, the process begins to denature and the DNA splits into 2 single-stranded DNA molecules for 30 seconds. Once the DNA is denatured, the DNA template is annealed where primers bind onto the DNA template. After that, the process begins to extend and the DNA polymerase is activated and it locates a primer attached to a single DNA strand and then starts to add complementary nucleotides. The extension process consists of four types of nucleotides (adenine (A), thymine (T), cytosine (C) and guanine (G)) where A pairs with T and G pairs with C. These four nucleotides are paired together by hydrogen bonding. This process allows primers to stick to the template strand. 30 seconds after the extension process, the process approaches the final step where the process continues to add nucleotides to strands and then the process cools down to 4° C (final hold) to conclude one cycle. Another cycle is repeated except using two DNA strands as templates. The diagram below summarizes this process:

Description of image


References

Kaiser, Gary. "MIcrobial Genetics." The Community College of Baltimore. The Community College of Baltimore, n.d. Web. 30 Oct. 2013. <http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNA/dna.html>.

"PCR Virtual Lab." Learn Genetics: Genetic Science Learning Center. The University of Utah, n.d. Web. 30 Oct. 2013. <http://learn.genetics.utah.edu/content/labs/pcr/>.

"What is PCR?." Open PCR. Open PCR, n.d. Web. 30 Oct. 2013. <http://openpcr.org/what-is-pcr>.

Greenwald, Ted, "DNA Sequencing For Fun And Profit: A Low-Cost Platform For Garage Biotech" Forbes.com, Web.




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