BME100 f2013:W900 Group12 L4

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BME 100 Fall 2013 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: Swaroon Sridhar:Open PCR Machine Engineer
Name: Swaroon Sridhar:
Open PCR Machine Engineer
Name: Christopher Lae:Open PCR Machine Engineer
Name: Christopher Lae:
Open PCR Machine Engineer
Name: Courtney Van Bussum:Protocol Planner
Name: Courtney Van Bussum:
Protocol Planner
Name: Nhi Nguyen:Protocol Planner
Name: Nhi Nguyen:
Protocol Planner
Name: Swathi Harikumar:Research and Development Specialist
Name: Swathi Harikumar:
Research and Development Specialist

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design


This is an image of a device called an Open PCR machine. An Open PCR Machine is a device that performs polymerase chain reactions in which DNA is modified by creating an environment that provides necessary temperature changes for this process to occur. The temperature changes consist of being heated and cooled for multiple cycles; the DNA present within the samples is separated when heated and then replicated when cooled, which in turn results in multiple copies from a single strand of DNA. The Open PCR machine can then be connected to the computer using the USB cable, which enables the user to control the number of cycles and temperatures for the experiment. With this information, the Open PCR Machine then will perform environmental changes on the DNA samples with the heating plate, heating lid, and cooling fan. Information is then processed by the circuit board, and transferred to the LED screen which displays the results.


Experimenting With the Connections

When we unplugged Part 3 from Part 6, the LCD display on the machine turned off and did not display anything.

When we unplugged the white wire that connects Part 6 to Part 2, the machine was unable to take temperature readings.

Image:PCR drawing.jpg

Test Run The machine was run on October 23, 2013 at 9:59AM. A major problem was encountered in that the machine (labeled Group 12) was stuck on cycle 1 during the whole test run.




Protocols

Thermal Cycler Program

Stage 1:

95°C for 3 minutes- DNA double helix unravels

Stage 2:

Denature; 95°C for 30 seconds- Two DNA strands separate further into two more DNA strands

Anneal; 57°C for 30 seconds- Primer attaches to specific DNA sequence

Extend; 72°C for 30 seconds- DNA polymerase attaches to primer and begins to add complementary nucleotides to template DNA and extend the chain

Stage 3:

72°C for 3 minutes- Polymerase extends the nucleotide chain to the complementary template DNA. Target sequence begins to form.

Stage 4:

Final Hold; 4°C- Allows for short term storage of the reaction by stopping the thermal cycling process


DNA Sample Set-up

Positive Control:

Cancer DNA template

Tube label: +

Patient 1 ID 39526

Replicate 1

Tube Label: +1A

Patient 1 ID 39526

Replicate 1

Tube Label: +1B

Patient 1 ID 39526

Replicate 1

Tube Label: +1C

Negative Control:

Cancer DNA template

Tube label: -

Patient 2 ID 31856

Replicate 1

Tube Label: -2A

Patient 2 ID 31856

Replicate 1

Tube Label: -2B

Patient 2 ID 31856

Replicate 1

Tube Label: -2C



DNA Sample Set-up Procedure

  1. Step 1: Label each of the eight test tubes with the appropriate identification label as indicated by the table above.
  2. Step 2: Using a micropipette, insert 50μL of the PCR reaction mix containing TAq DNA Polymerase, MgCl2 and dNTP's into PCR test tubes. Be sure that for each new test tube, a fresh micropipette tip is used to transfer the mix in order to avoid cross contamination. Each reaction mix will contain a different template DNA and will have a forward and reverse primer.
  3. Step 3: Place the reaction tubes into the thermocycler of the PCR machine.
  4. Step 4: Begin the thermocycler program at the respective heats and times to begin PCR process.


PCR Reaction Mix

- Taq DNA polymerase

- MgCl2

- dNTPs (deoxyribonucleotides A, T, C, and G)


DNA/ primer mix

-DNA sample from either Patient One or Patient Two

-Forward Primer

-Reverse Primer
(the same forward and reverse primers are present in each sample)

Research and Development

PCR - The Underlying Technology

Introduction to PCR and Components Polymerase Chain reaction also known as PCR is technique commonly used on laboratories in order to duplicate a certain DNA sequence and amplify the amount of DNA present. Making more DNA allows for the use of DNA in experiments, DNA profiling, research into bacterial and viral infections. The PCR reaction contains many components which allow the completion of the reaction. Various components allow for the duplicate of DNA such as primers, templates, polymerase, magnesium, deoxyribonuleotides. Template DNA is the original DNA which is used as a template to create the complementary strands of new DNA. Due to the semi-conservative property of DNA, the new DNA strands combine to create a duplicate of the original. Primers are short pieces of DNA that are designed to match the segments of DNA to copy. They are necessary in order for DNA polymerase to attach. DNA Taq Polymerase is a naturally-occurring complex of proteins whose function is to copy a cell's DNA before it divides in two. It attaches to primers and add nucleotides. Magnesium chloride as a catalyst for the PCR reaction. Magnesium in general acts as a co-factor for DNA polymerase. The reaction cannot proceed without the presence of magnesium. Deoxyribonucleotides are the building blocks of DNA. There are four types of nucleotides designated by the different bases adenine, thymine,cytosine and guanine. Adenine bonds with thymine and cytosine bonds with guanine.

Steps of PCR The steps of PCR allow for the replication of DNA to occur as effectively as possible. During the first step, the DNA samples are heated to 95 degrees Celsius for 3 minutes. This allows the DNA double helix structure to unravel into two separate strands. The second step is the denaturing of the DNA at the 95 degrees for 30 seconds. The hydrogen bonds between the complementary nucleotides are broken to separate the two strands of DNA. The next step is annealing which occurs at 57 degrees Celsius for 30 seconds. During this step, primers attach to specific DNA sequences on the template DNA strand. These primers bind to the exposed complementary sites. The complementary factor allows for the specific replication of the a sequence of DNA needed for research. The control over the types of primers allows for accuracy in DNA replication and credits the PCR process for being efficient and effective. The next step requires for the extension of the new DNA strand. This step occurs at 72 degrees Celsius for 30 seconds. During this step, DNA polymerase extends the nucleotides chain to the complementary template DNA strand. The target sequences also begin to form. The final step of the PCR process continues the extension of the new DNA strand. The DNA polymerase attaches to the primers and creates the new DNA strand of complementary nucleotides from the template DNA. This step also occurs at 72 degrees Celsius for 3 minutes. This PCR process cycle continues until enough copies of the target sequences is created in order of researches to perform experiments with. Each cycle produces double the DNA. The entire duration of the process is an hour to create billions of copies of DNA from a single strand of template DNA. This process is known as DNA amplification.

Image:PCRPicture.jpg

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