BME100 s2015:Group4 9amL4

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Owwnotebook icon.png BME 100 Spring 2015 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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OUR TEAM

Name: Measho Habtemichael
Role(s)
Name:Aaron Frisby
Role(s)
File:Cody manning.jpg
Name: Cody Manning
Role(s)
Name: Alexandra Torres
Role(s)
Name: Amanda Nguyen
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s
  • DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
  • A strip of empty PCR tubes
  • Disposable pipette tips: only use each only once. Never re-­‐use disposable pipette tips or samples will be cross-­‐contaminated
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G4 + Positive control none
G4 - Negative control none
G4 1-1 Patient 1, replicate 1 19902
G4 1-2 Patient 1, replicate 2 19902
G4 1-3 Patient 1, replicate 3 19902
G4 2-1 Patient 2, replicate 1 33338
G4 2-2 Patient 2, replicate 2 33338
G5 2-3 Patient 2, replicate 3 33338


DNA Sample Set-up Procedure

  1. Retrieve all materials
  2. In the eight patient tubes, ensure there is the PCR reaction mix
  3. Label each of the tubs according to the PCR Reaction Sample List
  4. In the negative control tube, add 50 microliters of negative control
  5. In the positive control tube, add 50 microliters of positive control
  6. Then in the three tubes for patient 1, add 50 microliters of patient 1's DNA mix
  7. In the three tubes for patient 2, add 50 microliters of of patient 2's DNA mix
  8. Close all of the tubes
  9. Place in OpenPCR machine

OpenPCR program

  • HEATED LID: 100°C
  • INITIAL STEP: 95°C for 2 minutes
  • NUMBER OF CYCLES: 35
  • Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds
  • FINAL STEP: 72°C for 2 minutes
  • FINAL HOLD: 4°C




Research and Development

PCR - The Underlying Technology

There are many components to a PCR reaction. A full reaction requires a DNA template, a DNA polymerase, primers, and nucleotides. The DNA template is a sample of DNA that contains the sequence that needs to be analyzed. This template is then manipulated in order to separate the strands of DNA from each other. DNA polymerase is an enzyme that creates complementary strands of DNA to the target sequence of DNA. The polymerase used in this reaction is Taq DNA polymerase. Primers are small pieces of single strands of DNA. These small pieces of DNA form together to create the complimentary strand of DNA. The polymerase then begins to create the new strands starting at the end of the primer. Nucleotides are the basic building blocks of DNA. They are the bases A, T, G, and C. These building blocks are used by the polymerase and primer in order to create the complementary strand of DNA.

PCRreaction.jpg
Source: http://www.ncbi.nlm.nih.gov/genome/probe/doc/TechPCR.shtml

During the INITIAL STEP, the DNA is heated to 95*C in order to begin the denaturing process. During the DENATURE step, DNA is heated even further in order to separate the double strands of DNA. The heat causes the hydrogen bonds within DNA to break, releasing the two strands. During the ANNEAL step, the temperature is lowered in order to allow the primers to adhere to the now single strands of DNA. During the EXTEND step, the temperature is increased slightly. At this temperature, the polymerase then begins working with the primers in order to begin to create complimentary strands of DNA. The FINAL STEP is to ensure that all complementary nucleotides have been attached to the complimentary strand. The FINAL HOLD is used for an indefinite period of time in order to maintain the complimentary strands of DNA until it is time for the strands to be used.

Adenine (A) bonds to Thymine (T)
Thymine (T) bonds to Adenine (A)
Cytosine (C) bonds to Guanine (G)
Guanine (G) bonds to Cytosine (C)