BME100 s2018:Group3 W1030 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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OUR TEAM

Name: John Le
Name: Mikayla Gardes
Name: Alicia Salas
Name: Anastasia Hancock

LAB 4 WRITE-UP

Protocol

Materials

  • Lab Coat & Disposable Gloves
  • PCR Reaction Mix, 8 tubes, 50 μL each
  • DNA/Primer Mix, 8 tubes, 50 μL each
  • A Strip of Empty PCR Tubes
  • Disposable Pipette Tips
  • Cup for Discarded Tips
  • Micropipettor
  • OpenPCR Machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G3 + Positive control none
G3 - Negative control none
G3 1-1 Patient 1, replicate 1 74852
G3 1-2 Patient 1, replicate 2 74852
G3 1-3 Patient 1, replicate 3 74852
G3 2-1 Patient 2, replicate 1 42241
G3 2-2 Patient 2, replicate 2 42241
G3 2-3 Patient 2, replicate 3 42241


DNA Sample Set-up Procedure

  1. Check to see if all materials listed in materials are present.
  2. Locate the empty PCR tubes and slice the strip in two so there are two strips of four tubes.
  3. Label the tubes with the group label that was created.
  4. Locate the rack and place the tubes inside of it.
  5. Find the empty positive control tube and fill it with 50 μL of the PCR reaction mix.
  6. Place the positive DNA/ primer mix in the positive control tube making the volume of the tube 100 μL.
  7. Repeat steps 5 and 6 for the other tube labels. There should be a corresponding DNA/ primer mix for each label. Each tube should have a volume of 100 μL.
  8. Cover the lids of the tubes.
  9. Take the tubes to the PCR machine. Locate the heating block and put the tubes inside of it. Only start the machine when all 16 spots are filled in the machine.


OpenPCR program

BME100 Group3 OpenPCR.PNG






Research and Development

PCR - The Underlying Technology

The PCR reaction has multiple components in order to make the reaction work. The components are Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides. The template DNA is a DNA sample that is used to target the specific DNA copies. The primers attach to the end of the DNA strand once the strands has been separated. Then they would copy the specific targeted DNA sequence. The taq polymerase reads the DNA sequence and translate it to match the matching the Nucleotides thus making a additional DNA copy. The Nucleotides are the codes that make up DNA strand.


The Components during the thermal cycling:

1. Initial Step (95°C for 2 minutes): The template DNA strands comes to temperature.

2. Denature (95°C for 30 seconds): The template DNA separates into two individual strands.

3. Anneal (57°C for 30 seconds): The primers attaches at the end of each DNA strand.

4. Extend (72°C for 30 seconds): The taq polymerase is activated and attached to the primers.

5. Final Step (72°C for 2 minutes): The polymerase begins to add complementary nucleotides on DNA stands. Which replicates the targeted DNA.

6. Final Hold (4°C): Copies are made.


DNA is made up of four types of mucleotides and the are put together to create a strand. Adenine is paired with Thymine and vice versa. Cytosine is paired with Guanine and vice versa. This is essentially the basic building blocks of DNA.

The main cycle that base pairing occurs is in the Anneal and Extend Cycle because that is when the taq polymerase is activated and attached to the primers. Once the primers and taq polymerase are attached, they begin to add complementary nucleotides to make a copy of the template DNA.




SNP Information & Primer Design

Background: About the Disease SNP The species that this variation is found in is Homo Sapiens, and it is located on the 6:131851228 chromosome. This variation has been linked to metabolic diseases. ENPP1 is the ectonucleotide pyrophosphate/ phosphodiesterase, and its function is for zinc ion binding, insulin receptor binding, and protein binding. The disease-associated codon is CAG. It is located at 131851228.


Primer Design and Testing

Lab 4 Pic.PNG