Name: Chloe Houlihan Role(s): Research and Development
Name: Brianna Snyder Role(s): Machine Testing
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The OpenPCR machine is a major facet of this experiment. Though not as efficient as the medical standard issue PCR machines such as the Mastercycler used by insurance agencies, but it certainly gets the job done. The machine we used has all the capabilities as the professional models in that the OpenPCR machine has a dual power circuit that allows for emergency back-up. Additionally, a cooling fan and a power fan are included to ensure that external heat measurements are not a major source of error.
Experimenting With the Connections
When we unplugged the LCD screen from the PCR Brains Board, the LCD screen turned off.
When we unplugged the white wire that connects the PCR Brains Board to the 16-well PCR block , the machine read that the internal energy went from 25 degrees Celsius to -40 degrees Celsius.
Test Run
The test run was performed on March 19. It began at 10:08 am. The test ran for an hour and performed 18 cycles. The PCR machine passed.
Protocols
Thermal Cycler Program
DNA Sample Set-up
Materials
8 PCR tubes (empty and attached)
PCR reaction mix in 8 PCR tubes
8 Tubes of Template DNA/Primer Mix, 50μl
Disposable pipette tips
200μl Micropipettor
Surgical gloves
DNA Sample Set-up Procedure
Step 1: Cut strip of empty PCR tubes in half. This way you will have two strips of 4 empty PCR tubes.
Step 2: Use a black marker to label the tubes.
Step 3: Place tubes in a rack.
Step 4: Start with an empty tube labeled as a positive control.
Step 5: Transfer 50μl of PCR reaction mix to an empty tube.
Step 6: Transfer +DNA/Primer mix into the tube with PCR reaction mix.
Step 7: Repeat step 5 and step 6 with the negative control.
Step 8: Close all lids tightly and insert the tubes into the OpenPCR machine.
PCR Reaction Mix and DNA/primer mix
Inside the PCR master reaction mix there are six different components. The template, the primers, a DNA polymerase, the MgCl2 concentration, Deoxynucleotide triphosphate (dNTP), and lastly is the pH of the solution. Inside the DNA/ primer mix are the template strand of DNA that is going to be replicated, and the DNA polymerase that once active will construct the new DNA strand. The primer mix is an enzyme that attaches to the first three nucleotides in a DNA base.
Research and Development
PCR - The Underlying Technology
The Basics
A nucleotide is the basic building block for nucleic acids, and consists of a nitrogenous base, a sugar, and a phosphate group. A polymorphism is when two or more different phenotypes exist in a population. An allele is one of a number of alternative forms of the same gene.
The DNA
One single nucleotide polymorphism (SNP) is rs237025. This SNP is associated with the SUMO4 gene and is found in Homo sapiens. SUMO4 stands for small ubiquitin-like modifier 4. It attaches to proteins and controls the target proteins' subcellular localization, stability, or activity. It is located on the chromosome 149721690. Type I and Type II diabetes as well as rheumatoid arthritis are linked to this SNP. The disease-associated allele contains the sequence ATG. This SNP occurs at the location 149721690. Exactly 200 bases to the right is the numerical position 149721890.
The DNA Sequence
The image above shows the forward primer for the disease-allele.
The image above shows the reverse primer for the disease-allele.
Will It Work
PCR will not happen if the template contain the non-disease-allele because the disease-allele primer will not be able bind to the template strand. Crossing SNPs of a non-disease and a disease SNP means that they are not compatible and that the PCR reaction will essentially fail.
BME 100 Spring 2014
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