Name: Parker Davis Role(s)Research and Development
Michael Spina Role(s)Protocols
Name: Brian Kalen Role(s)Initial Machine Testing
Name: Daniel Munoz Role(s)Research and Development
Name: Rakan Aldrssary Role(s)Protocols
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The OpenPCR machine consists of thin pieces of wood, and a case that contains the hardware of the machine. A second, wood case on top of the main body contains the place where the samples are put into the machine. Small test vials are placed in the holders in the loading area to be heated by the PCR machine. Inside the main body of the machine there is hardware similar to a computer. A processor unit connects to the heating device as well as the power supply and LED screen. Above the power supply there is a heatsink, with a fan attached, facing some vents inside the machine, which allows for efficient temperature control inside the machine to prevent overheating. The machine then has components in order to be hooked up by USB to a computer. With the computer the PCR can be programmed to heat up the samples in the heating lid on certain time intervals, different temperatures, and indicated cycles.
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine
When part 3 was removed from part 6 the LED display screen shut off.
When we unplugged the white wire that connects (part 6) to (part 2), the machine
The temperature reading turned negative.
Test Run
The group first tested the machine on March 20, 2014. The first running of the machine went well, but the machine worked very slow. The 35 cycles took well over the two hour mark and the run was not able to be finished on time with class ending. This required us to fail the machines run.
Protocols
Thermal Cycler Program
DNA Sample Set-up
row 1 cell 1
row 1 cell 2
row 1 cell 3
row 1 cell 4
row 2 cell 1
row 2 cell 2
row 2 cell 3
row 2 cell 4
C+
p11
p12
p13
C-
p21
p22
p23
DNA Sample Set-up Procedure
Step 1 Obtain the PCR reaction and DNA primer mix
Step 2 Mix the reaction mix and primer mix into one tube
Step 3 Put the mixes into the tubes to be inserted into the machine
Step 4 Run PCR via computer
PCR Reaction Mix
What is in the PCR reaction mix?
The reaction mix contains Taq DNA polymerase, MgCl2, and dNTP's.
DNA/ primer mix
What is in the DNA/ primer mix?
The primer mix contains a different template DNA. All tubes have the same forward and reverse primer.
Research and Development
PCR - The Underlying Technology
Basic concepts
DNA is the blue print of the human being. DNA is made up of nucleotides and peptide bonds. These four types of nucleotides form long strands to make up entire genomes. The human genome is composed of every gene that makes up every trait a human can have. Phenotypes are physically expressed by a human. Phenotypes are things like hair color or skin tone. Each type of phenotype has the possibility of being present in a population. When two or more phenotypes are present in the same population, this is known as polymorphism.
Specifics on polymorphism
In humans there are many different examples of polymorphism. For example, the single nucleotide polymorphism known as RS237025 is associated with the phenotypes of diabetes. The polymorphism that codes for this can tell if a person will either get type 1 diabetes or not. This polymorphism is associated with the gene SUMO4, or Small Ubiquitin-Modifier 4. This gene is located in the cytoplasm and modifies the protein IKBA.
The allele of polymorphism
Each polymorphism is due to two or more different alleles which code for the same gene.
In this case the allele that is associated with the gene that codes for type 1 diabetes contains the sequence ATG while the non diseased allele contains the sequence GTG. The change of one base determines whether the person in question will have type 1 diabetes or not.
BME 100 Spring 2014
