BME100 s2014:W Group6 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

GROUP 6

Elizabeth BurkettRose Among the Thorns
Elizabeth Burkett
Rose Among the Thorns
Matthew WarrenThe Handy Man
Matthew Warren
The Handy Man
Samuel McCollumMan in Charge
Samuel McCollum
Man in Charge
Frederick SebastianWetWare Master
Frederick Sebastian
WetWare Master

LAB 4 WRITE-UP

Initial Machine Testing

The Original Design
Image:Group_6_pcr.JPG

This device called Open PCR, is a machine that can polymerase chain reactions to separate a DNA strand and use one strand to create a complimentary strand. This results in a new double strand DNA molecule, that is initially identical to the first DNA molecule.

This is made possible by the machine heating the test tubes with primer 1, primer 2, polymerase, nucleotides, DNA, and DNA polymerase ad also subsequently cooling them. These heating and cooling procedures with the various contents in the test tubes creates an environment that allows for this DNA molecule replication.

The machine is run by connecting it to a computer using a USB cable and running a program that tells the machine the specifics of the desired experiment. Like the number of cycles and temperature to be run at. The machine is also hooked on its own power supply to carry out these processes. Once the experiment is done, the machine displays the results on the blue screen on the machine.


Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine did not display the necessar information on the blue screen that's on top of the thermocycle.

When we unplugged the white wire that connects (part 6) to (part 2), the machine the blue display screen wold not display the temperature of the PCR test tubes

Test Run

March 19th, 2014.

The internal components of the machine was tested to see if they are in working conditions, and to ensure that there is no malfunction. The machine was then loaded with empty test tubes to create a sample run using the software on the computer to ensure the mother board on the machine works properly. The machine run was simple and easy to be done since the required information for the run was already given and the internal components of the PCR were not faulty. After the machine completed its test run (16 minutes and 30 cycles) we concluded that the machine passed the test.




Protocols

Thermal Cycler Program

  • Test tubes with primer 1, primer 2, polymerase, nucleotides, DNA, and DNA polymerase are heated at 95°C for 3 minutes
  • The contents denature at 95°C for 30 seconds. Double strands of DNA separate.
  • The contents then anneal at 57°C for 30 seconds. Primers attach at ends of target DNA segment.
  • The contents then extend at 72°C for 30 seconds. DNA polymerase activates and replicates target segment of DNA.
  • The test tubes remain at 72°C for 3 minutes
  • The test tubes are then cooled to 4°C, then the PCR reaction is stopped.


DNA Sample Set-up

The two patient ID's are 67618 and 78379 respecively. The positive control is labelled (PC) and the negative control is labelled (NC). Patient 1's samples are labelled P11, P12, and P13. In a similar manner Patient 2's samples are labelled P21, P22, P23.

PC P11 P12 P13
NC P21 P22 P23


DNA Sample Set-up Procedure

  1. 8 test tubes each containing 50μL of PCR reaction mix are prepared
  2. The test tubes are labelled according to the table above
  3. 50μL of each DNA sample Mix was added to the labeled test tubes. We used a new pipette tip for each transfer in order to avoid cross-contamination between samples.
  4. The test tubes are placed into the OpenPCR machine
  5. The thermocycler process is then started

PCR Reaction Mix

  • 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s.


DNA/ primer mix

  • 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.





Research and Development

PCR - The Underlying Technology
The underlying technology that enables PCR is the thermocycler. This device allows one to control when and to what degree the temperature is changed. Everything else is controlled by the reagents present in the reaction tube. The combination of these two factors allows for the replication of the existing DNA.

Component functions

The template DNA is the strand of DNA from which another is copied. Primers allow the DNA polymerase to attach to the DNA strand. Taq Polymerase adds nucleotides to the template DNA strand and provide beginnings and ends. Deoxyribonucleotides are added to the template stands of DNA to form a new strand of anti-parallel DNA.

Changes made to the components during thermocycle

The initial step will occur for 3 minutes at 95ºC. This step will heat the solution to the desired 95ºC. The next step will be to denature the DNA for 30 seconds at 95ºC. This will allow for the 2 DNA strands to separate. The next step is to anneal the DNA for 30 seconds at 57ºC. In this step the primers attach to the DNA strands before the strands rejoin. Next the extension period will last for 30 seconds at 72ºC. In this step the polymerase is activated, causing the nucleotides to be added to the strand. The final step will last for 3 minutes at 72ºC, which prepares the hot plate further and ensures accuracy. Lastly, the final hold will occur at 4ºC and will stabilize the DNA.

DNA structure

DNA is made up of four types of molecules called nucleotides, designated as Adenine (A), Thymine (T), Cytosine (C) and Guanine (G). base-pairing, which is driven by hydrogen bonding, will allow the DNA strands to stick together. This will also allow primers to stick to the template DNA strand. Adenine will always pair with Thymine, and Cytosine with Guanine. For example, if a DNA sequence is ATGAC, the complimentary base pair would be TACTG.




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