BME100 s2014:W Group12 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: RJParkinson
Name: RJ
Parkinson
Name: SaraJerez
Name: Sara
Jerez
Name: AustinDoyle
Name: Austin
Doyle
Name: AnthonyZlaket
Name: Anthony
Zlaket
Name: JacquelineStokes
Name: Jacqueline
Stokes

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:OpenPCR.jpg

This OpenPCR machine performs the PCR process. The first step is to heat up the micro test tubes containing a sample of DNA to 95 degrees Celsius. The process continues by separating the double helix into two separate strands. The primer is attached to the end of each strand, then the polymerase attaches and copies the second half of each strand, forming replicates of the template DNA. The process ends by having the machine cool down the temperature to 4 degrees Celsius. This machine duplicates DNA for the purposes of detecting viral and bacterial infections, genotyping, food safety testing, and scientific research.


Experimenting With the Connections

When we unplugged (part 3) from (part 6), the monitor turned off but the rest of the machine was still functioning.

When we unplugged the white wire that connects (part 6) to (part 2), the temperature on the monitor changed from 25.6 degrees Celsius to -40 degrees Celsius. This wire connects the thermometer to the circuit board, therefore when we unplugged it the actual temperature could not be displayed on the monitor.

PASS. The temperature displayed on the monitor was increasing and decreasing throughout the test run, from 95 degrees Celsius to roughly 50 degrees Celsius. Out of 35 cycles, it reached 23 within about an hour of time.


Test Run

After placing the samples in the machine and setting up the program on one of our computers we started the PCR machine and went through several cycles. While the machine was running we periodically checked that the temperature readings on the machine corresponded with the readings displayed on the computer and found that they were consistently correlated. We did not go through all 35 of the cycles in the interest of time, but based on the fact that the first 23 cycles went through without any problems we believe that the machine would have gone through the rest of the cycles without a hitch.




Protocols

Thermal Cycler Program


DNA Sample Set-up

RJ1 RJ2 RJ3 RJ4
RJ5 RJ6 RJ7 RJ8


DNA Sample Set-up Procedure

  1. Step 1: Identify a positive and negative control
  2. Step 2: Gather the materials for the PCR process, (the PCR reaction mix and DNA/primer mix)
  3. Step 3: Micropipet equal amounts of PCR reaction mix and DNA/primer mix (or the positive/negative controls) into the micro test tubes.
  4. Step 4: Begin the PCR process.


PCR Reaction Mix

  • What is in the PCR reaction mix?

This includes the polymerase molecules.


DNA/ primer mix

  • What is in the DNA/ primer mix?

This is a mixture of the DNA primers.





Research and Development

PCR - The Underlying Technology

Components of the PCR reaction

Within the PCR mix there are several components which all contribute to the amplification of DNA. Within the mixture is the original sample of template DNA, this strand of DNA is the original sample that is going to be copied several times over. In addition to the DNA several DNA primers are added to the mixture. These primers attach to the single stranded DNA and mark the binding sites for the Taq Polymerase molecules. The Taq Polymerase molecules are the structures that actually bind complimentary nucleotides to the DNA strand turning the DNA into a double strand. Deoxyribonucleotides are the basic unit of the DNA molecule consiting of a nucleic acid, a sugar base and a phosphate backbone. As the Taq Polymerase moves down the strand of DNA deoxyribonucleotides (dNTP's) are binded to the single strand to create a new double strand of DNA.

The Thermocycling Process

Initially the DNA is placed in the machine and heated to 95 degrees Celsius of 3 minutes in order for the machine to heat up to begin the PCR process.Then the DNA is kept at 95 degrees Celsius this step denatures the DNA molecule, breaking the hydrogen bonds keeping the two strands together resulting in two single strands of DNA. After denaturing the DNA the mixture is brought down to 57 degrees Celsius for the annealing stage, here the DNA primers bond to the single stranded DNA in preparation for the Taq molecule. Then the mixture is heated to 72 degrees Celsius for the extending stage. Here taq Polymerase bonds to the primers and begins to add complimentary dNTP's to the single strand of DNA. Then the mixture is held at 72 degrees Celsius for 3 minutes in order for the polymerase to have enough time to complete the process of adding dNTP's. Then the mixture is held at 4 degrees Celsius in order to complete the PCR cycle and stabilize the DNA molecules.





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