BME100 f2013:W1200 Group1 L4

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Contents

OUR TEAM

Name: Chloe KwonRole: Research and Development
Name: Chloe Kwon
Role: Research and Development
Name: Nima AfzalianRole: Open PCR Machine Testing
Name: Nima Afzalian
Role: Open PCR Machine Testing
Name: Casey WeinsteinRole: Open PCR Machine Testing
Name: Casey Weinstein
Role: Open PCR Machine Testing
Name: Jeff MilesRole: Protocol Planning
Name: Jeff Miles
Role: Protocol Planning

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

 Image:Group1PCRsetup.jpg


Experimenting With the Connections

The PCR machine is heavily dependent on numerous connections and and interconnected parts. This means that, in order for it to work well, everything must be in its proper location and plugged in. This being said, when we unplugged part 3 (the LCD screen) from part 6 (circuit board) of the machine, the connection from the LCD to the circuit board was severed, resulting in the screen turning off. Also, when we unplugged the white wire that connects part 6 (circuit board) to part 2 (core) of the machine, the temperature changed from 26.7 degrees Celsius to -40 degrees Celsius. It is very important that all connections are correct in order to have an effective PCR machine.

Test Run
Our group's PCR machine was tested on October 23. The test began at 1:08 pm. After 30 minutes, 8 cycles were completed.




Protocols

Thermal Cycler Program

Initial Step

95°C for 3 minutes: The initial Template DNA strand separates

Denature

95°C for 30 seconds: The DNA double helix separates, creating two single-stranded DNA molecules

Anneal

57°C for 30 seconds: The two primers attach to opposite ends of the top and bottom strand of the target DNA segment

Extend

72°C for 30 seconds: The DNA polymerase activates. Replication of the target DNA segment begins.

Final Step

72°C for 3 minutes: The complementary binding of nucleotides continues until it gets o the end of the DNA strand and falls off.

Final Hold

4°C: The PCR reaction ends.

Image:group1PCRprogram.jpg



DNA Sample Set-up

Tube A: Positive Control: cancer DNA template Tube B: Patient 1 ID-16946 Replicate 1 Tube C: Patient 1 ID-16946 Replicate 2 Tube D: Patient 1 ID-16946 Replicate 3
Tube E: Negative Control: non-cancer DNA template Tube F: Patient 2 ID-46296 Replicate 1 Tube G: Patient 2 ID-46296 Replicate 2 Tube G: Patient 2 ID-46296 Replicate 3


DNA Sample Set-up Procedure

  1. Step 1: Fill each reaction tube with 50μL of the PCR reaction mix and label all tubes according to the chart above
  2. Step 2: Add 50μL of DNA sample mix to the corresponding reaction tube (Note: use each disposable pipette tip only once to avoid cross-contamination)
  3. Step 3: Place to reaction tubes into the thermocycler and run the above program


PCR Reaction Mix

The PCR reaction mix will contain 8 tubes. Each tube will contain 50μL of the following:

  • Taq DNA polymerase
  • MgCl2
  • dNTP's


DNA/ primer mix

The DNA primer mix contains 50μL of the following:

  • an extracted sample of the patient's DNA
  • forward primer
  • reverse primer

Research and Development

PCR - The Underlying Technology

(Add a write-up, essay-style, organized into paragrpahs with descriptive headers, based on the Q&A's from Section three of your worksheet)

(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 PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)





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