BME100 s2014:T Group4 L4

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BME 100 Spring 2014 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: Parker DavisRole(s)Research and Development
Name: Parker Davis
Role(s)Research and Development
Michael SpinaRole(s)Protocols
Michael Spina
Role(s)Protocols
Name: Brian KalenRole(s)Initial Machine Testing
Name: Brian Kalen
Role(s)Initial Machine Testing
Name: Daniel MunozRole(s)Research and Development
Name: Daniel Munoz
Role(s)Research and Development
Name: Rakan AldrssaryRole(s)Protocols
Name: Rakan Aldrssary
Role(s)Protocols

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:unamed.jpg


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

  1. Step 1 Obtain the PCR reaction and DNA primer mix
  2. Step 2 Mix the reaction mix and primer mix into one tube
  3. Step 3 Put the mixes into the tubes to be inserted into the machine
  4. 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.




Bonus
See Images Below
How Primers Bind to DNA Template
Image:Primer_Binding.jpg
picture found at: http://pulpbits.com/6-dna-semi-conservative-replication-animation/dna-replication-semiconservative-images/

How Taq Polymerases Amplify the DNA
Image:DNA_Amplification.jpg
picture found at: http://mistralmtn.blogspot.com/2012/12/pcr-technology-that-speeds-dna-tests.html





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