BME103 s2013:T900 Group4

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Lab Write-Up 1
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Lab Write-Up 3
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Contents

OUR TEAM

Name: Kinjal AhirRole: Experimental Protocol Planner
Name: Kinjal Ahir
Role: Experimental Protocol Planner
Name: Zach YoungRole: Initial Machine Testing
Name: Zach Young
Role: Initial Machine Testing
Name: Anna EssexRole: Initial Machine Testing
Name: Anna Essex
Role: Initial Machine Testing
Name: Amelia LaxRole: Research and Development
Name: Amelia Lax
Role: Research and Development
Name: Tuan PhanRole: Research and Development
Name: Tuan Phan
Role: Research and Development

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
(Add image of the full OpenPCR machine here) Here's the OpenPCR machine. Made up of thin pieces of wood, the case contains the hardware of the machine. A second, smaller wood case on top of the main case contains the loading bay, which acts as a lid. Small test vials can be placed into holders here in the bay to be heated by the PCR machine. Inside the main body of the machine sits the hardware similar to a personal computer. A processor unit connects to the heating hardware as well as the power supply and LED display board. Above the power supply lies the heatsink, with a fan attached, facing vents in the case, which allows for efficient thermal control inside the machine to prevent overheating. The machine then has sockets that allow the machine to be hooked up by USB to a computer. Using a computer the PCR can be ordered to heat up the vials in the heating lid on certain intervals at different temperatures as well as indicated cycles.


Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer)

When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer)


Test Run

(Write the date you first tested Open PCR and your experience(s) with the machine)




Protocols

Thermal Cycler Program

DNA Sample, 50 μL each: patient ID's from the UnderGrad Teaching Assistant(UGTA).

Positive control Patient 1 Patient 1 Patient1
cancer DNA template ID: 85158 ID: 85158 ID: 85158
Replicate 1 Replicate 2 Replicate 3
Tube label: A+ Tube label: B1 Tube label: C1 Tube label: D1
Negative Control: no Patient 2 Patient 2 Patient 2
DNA template ID: 17818 ID: 17818 ID: 17818
Replicate 1 Replicate 2 Replicate 3
Tube label: A- Tube label: B2 Tube label: C2 Tube label: D2

DNA Sample Set-up Procedure

  1. Step 1: Student were Provided with 8 tubes of 50 μL PCR reaction mix, 8 tubes of 50 μL diluted template + primers, and disposable transfer pipettes.
  2. Step 2: Protocol planner labeled the test tube that is provided above.
  3. Step 3: 50μL of each DNA sample Mix in to test tube.
  4. Step 4: Protocol planner set up the DNA and ran the PCR reactions.


PCR Reaction Mix

  • In the PCR reaction mix there is Mix contains Taq DNA polymerase, MgCl2, dNTP's, forward primer, reverse primer.

DNA/ primer mix

  • In the DNA/ primer mix there is patient's DNA.






Research and Development

Specific Cancer Marker Detection - The Underlying Technology

First the DNA/Primer mix is combined with the PCR reaction mix in a small test tube. Repeat samples are advised to make sure that the results are consistent.

When the test tube is placed in a PCR machine, the mixture is heated to 95 degrees Celsius for 30 seconds. This high heat breaks apart complementary strands of DNA, exposing the nucleotides. The DNA strands are essentially cut in half lengthwise into two new strands.

The machine then rapidly cools to 57 degrees Celsius for 30 seconds, allowing the DNA primers to bind to the exposed sites. The DNA primers can only bind to the specific site they are complementary to, which allows researchers to target specific DNA sequences. Even if only one nucleotide is different, the primer does not bind well enough to stay. This means that researchers can choose a specific gene, such as one predisposing for cancer, and order a primer that binds only to that gene. If the specified gene is not present, the primer will not bind and the DNA will not be amplified.

Two different primers are needed: a forward primer and reverse primer. The forward primer binds to one "half" of the DNA sample, and the reverse primer binds to the other "half." If you picture the DNA as a train track, with each rail as one half of the DNA, one primer binds to one rail, and the other binds to the other rail at the opposite end of the track.

The mixture is then heated up to 72 degrees Celsius for 30 seconds. At this temperature, DNA polymerase binds to the primers and builds a new segment of complementary DNA with the free nucleotides in the mixture. Returning to the train track analogy, one rail is being built from right to left and the other from left to right, both being built towards each other. This is because DNA is read in one direction on one side, and a different direction (relative to the first) on the other.

So, if there was only one strand of DNA in the mixture originally, now there are two (since both halves have been replicated). The mixture is then reheated to 95 degrees and the cycle continues, each time doubling the amount of DNA in the mixture. This process usually continues for an hour or so, until there are billions of copies of DNA. This whole process is referred to as DNA amplification.


(Add a write-up of the information discussed in Week 3's class)

(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)





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