BME100 s2015:Group7 9amL4

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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: Luke ShafferRole(s)
Name: Luke Shaffer
Role(s)
Name: Brandon HansenRole(s)
Name: Brandon Hansen
Role(s)
Name: Harris TranRole(s)
Name: Harris Tran
Role(s)
Name: Jaid CoronelRole(s)
Name: Jaid Coronel
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab Coat
  • Disposable Gloves
  • PCR reaction mix
  • 8 tubes, 50 μL each contains Taq DNA polymerase, MgCl2, and dNTP’s
  • 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
G7 + Positive control none
G7 - Negative control none
G7 1-1 Patient 1, replicate 1 71504
G7 1-2 Patient 1, replicate 2 71504
G7 1-3 Patient 1, replicate 3 71504
G7 2-1 Patient 2, replicate 1 96389
G7 2-2 Patient 2, replicate 2 96389
G7 2-3 Patient 2, replicate 3 96389


DNA Sample Set-up Procedure

  1. Check that group has all required materials
  2. Cut strip of empty PCR tubes in half, resulting in two strips of four linked tubes
  3. Label the sides of the empty tube with tube labels, making sure to not mark the lids
  4. Place tubes in rack
  5. Transfer 50 μL of PCR reaction mix into the tube labeled positive control, disposing of the tip into the collection cup afterwards
  6. With a new pipette tip, transfer the positive control DNA/primer mix into the same tube.
  7. Repeat steps 5 and 6 for the negative control, patient 1 replicates 1,2, and 3, and patient 2 replicates 1,2, and 3.
  8. Seal the lids of the PCR reaction tubes tightly.
  9. Take the tubes to the PCR machine, and place them into the heating block.
  10. Return all reusable materials to the appropriate storage places.
  11. Dispose of all biomedical material waste in an orange/ red Biohazard Bag.


OpenPCR program

The thermal cycling program is a program that is used to control the OpenPCR machine. Using a varied set of input data, the program can tell the machine to heat up to a specific temperature, cool down to a specific temperature, and specify the number of times to repeat the cycle.





Research and Development

PCR - The Underlying Technology


The purpose of the PCR (Polymerase Chain Reaction) is to replicate a small strand of DNA sample into a batch large enough to measure through technological means. The DNA sample taken from a patient acts as the template for the replication to base from; meaning after the process of the chain reaction, multiple amounts of the patient's DNA is measurable. While primers in the chemical solution acts as the beginning placeholders for Taq polymerase to add complementary nucleic acids (such as Adenine, Thymine, Cytosine, Guanine; binding to Thymine, Adenine, Guanine, Cytosine respectively) to the DNA template. The deoxyribonucleotides (dNTP's) acts as the building materials in this solution, for the polymerase to attach onto the separated DNA strand. These are the main components for PCR to perform effectively and efficiently.

During the PCR process, a cultivating machine heats up the DNA smaple mixture; essentially accelerating the process, which normally proceed slowly without any energy-incentive. The initialization step of the process begins this procedure (368K for 3 min). Next, the denaturation step separates the DNA template within the mixture into single strands by disrupting the hydrogen bonds between each paired bases (368K for 30 sec). Afterwards, the temperature lowers to 330K (30 sec) to allow primers to anneal to each patient's individual strands of DNA. This primes the replication process of the PCR procedure. It is by the end of the annealing step that Taq polymerase begins to extend, or elongate, upon each individual DNA strand--pairing free-roaming dNTPs to complementary nucleic bases and synthesizing new pair of the patient's DNA (345K for 30 sec). It is crucial around this time for the DNA strands to form; if the temperature is too high both the primer and the bases cannot bond to their respective region, and if the temperature is too low either may not properly bond.

As soon as the elongation are complete, the PCR cycles multiply times (from denaturating to elongation) until a desired amount of the sample is achieved. From here, the machine proceed with the final elongation (345K for 3 min)--to ensure any remaining individuals to be synthesize into full DNA strands--then cools to 277K for the final product to be used or stored.

Above is a hand drawn representation of the DNA as it goes through the denaturation step of PCR
Above is a hand drawn representation of the DNA as it goes through the denaturation step of PCR




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