BME100 f2013:W1200 Group14 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

OUR TEAM

Name: Vaasavi Sundar  Research and Development
Name: Vaasavi Sundar
Research and Development
Name: Brie Schilling  Open PCR Machine Testing
Name: Brie Schilling
Open PCR Machine Testing
Name: Minh Pham  Open PCR Machine Testing
Name: Minh Pham
Open PCR Machine Testing
Name: Ashley Powell  Protocol Planning
Name: Ashley Powell
Protocol Planning
Name: Hannah Brutsche  Protocol Planning
Name: Hannah Brutsche
Protocol Planning

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
OpenPCR is a device used by scientists to copy specific sections of DNA strands and amplify them through a Polymerase Chain Reaction (PCR). DNA strands are separated within the machine through a series of heating and cooling cycles. With the addition of polymerases, complementary strands to certain segments of the DNA are copied multiple times within a matter of hours from an original strand of DNA. Through this process, specific segments are amplified to be observed so one can find and diagnose diseases, viruses, or bacteria. The samples inside the heating plate experience all the different conditions. The Open PCR machine is connected through a USB cord to a computer, which through the OpenPCR software controls the heating and cooling cycles that the DNA samples undergo. The USB connection to the computer allows one to alter the experiment to reach fixed values and observe the changes within the sample.
Image:Pcr_photo.JPG

Experimenting With the Connections

When we unplugged the LCD screen (part 3) from the circuit board(part 6), the display screen turns off and shows nothing at all.

When we unplugged the white wire that connects the circuit board(part 6) to the heating plate(part 2), the temperature sensor malfunctions and displays the incorrect temperature on the LCD screen.


Image:Pcr111.jpg

Test Run

The test was run on Wednesday, October 23rd, 2013 at 1:08 PM.




Protocols

DNA Sample Set-up

Positive Control: Cancer DNA Template,
Tube Label: CT
Patient 1
ID: 30337
Replicate 1
Tube Label: 303
Patient 1
ID: 30337
Replicate 2
Tube Label: 304
Patient 1
ID: 30337
Replicate 3
Tube Label: 305
Negative Control: Non-Cancer DNA Template,
Tube Label: NCT
Patient 2
ID: 57707
Replicate 1
Tube Label: 577
Patient 2
ID: 57707
Replicate 2
Tube Label: 578
Patient 2
ID: 57707
Replicate 3
Tube Label: 579

Materials
PCR Reaction mix, 8 tubes (50 micrometers), DNA primer mix, disposable pipette


DNA Sample Set-up Procedure
1) Label all different tubes so we don't mix up any of the results.
2) Add 25μL of DNA sample mix to each corresponding tube, using different disposable pipette tips for each tube.
3) Place the tubes into a thermocycler
4) Program the Thermocycler to run as follows so PCR occurs.

Thermocycler Program
1. 1 Cycle at 95°C for 3 minutes
2. 35 Cycles at 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds
3. 72°C for 3 minutes
4. Hold the mix at 4°C

Image:Screenshot of open pcr software.jpg
PCR Reaction Mix
The PCR Reaction mix has Taq DNA Polymerase, Magnesium Chloride, and deoxynucleotide triphosphates.


DNA/ primer mix
DNA synthesis is when you make two identical copies of a molecule of DNA from a single original molecule. The primer is the beginning point for the DNA synthesis process. It is a strand of nucleic acids that accepts new nucleic acids to match the strand being copied. The DNA primer mix has the same forward primer and reverse primer for all tubes and a different template DNA in each tube.





Research and Development

PCR - The Underlying Technology


Components of PCR:

PCR, or polymerase chain reaction, is used to amplify DNA to diagnose disease, map the human genome, or even to solve crimes. There are several components involved in completing PCR-- the template DNA, the primers, the Taq polymerase, magnesium chloride, and deoxyribonucleotides. Through a process of heating and cooling, the DNA is rapidly amplified. Usually, there is a target sequence within the template DNA that needs to be amplified-- through the heating and cooling process, the target sequence is amplified. The purpose of each component is as follows:

Template DNA: The template DNA contains the target sequence, and is the basis for the amplification. Without the template, the target sequence cannot be amplified, and the primers have nothing to bind to, and polymerase has nothing to copy.

Primers: The primers bind to each single-stranded DNA and functions almost as a signal for Taq polymerase to begin the construction of the complementary sequence. Furthermore, the presence of both forward and reverse primers ensures that the target sequence, in particular, will be amplified, because the primers are made specifically for the sections of the template DNA which include the target sequence.

Taq polymerase: This is an enzyme which constructs the complimentary sequence to the single strands of the template DNA.

Magnesium chloride: The magnesium chloride acts as a catylyst for the polymerase reaction, and helps the reaction move along faster.

Deoxyribonucleotides: These are free-floating nucleotides, and these are the bases that are used by Taq polymerase to create the complimentary sequence for the single-stranded DNA. These nucleotides are adenine, guanine, thymine and cytosine. Adenine is paired with thymine, cytosine with guanine.

PCR Cycle:

The aforementioned materials are the key components in a PCR reaction. The first step of PCR is to heat the DNA to around 95 degrees celsius for around 3 minutes, and this allows the DNA to denature and unwind into two separate strands. The temperature, which is near boiling, and the required time, are both imperative to this reaction because this is the amount of energy that is required to break the hydrogen bonds between the nucleotides of the complementary strands. Once the DNA denatures, the temperature must be lowered to 57 degrees for around 30 seconds. The lowering of temperature allows for the forward and reverse primers to bind onto each single stranded-DNA. After 30 seconds of this, the temperature is once again re-heated to 72 degrees, the ideal temperature for Taq polymerase. Taq polymerase now begins constructing complementary strands to each single template strand. This continues for around 3 minutes. This cycle repeats itself over and over, until the "target sequence" is amplified more so than the original template DNA. The strands are complementary, and are created with paired nucleotides-- adenine binds with thymine, and cytosine with guanine. Base pairing, which is done through hydrogen bonds, are what create the "spiral staircase" of DNA structure. This is also what allows primers to the template strand.





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