BME100 f2013:W1200 Group9 L4

From OpenWetWare

Jump to: navigation, search
BME 100 Fall 2013 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help
Image:BME494_Asu_logo.png


Contents

OUR TEAM

Image:.jpg
Name: Jessica Stradford
Role(s): Open PCR machine testing
Image:.jpg
Name: Rachael Hall
Role(s): Open PCR machine testing
Image:.jpg
Name: Kenna Lum
Role(s): Protocol Planning
Image:.jpg
Name: Michelle Sigona
Role(s): Research & Development
Image:.jpg
Name: Salvador Avina
Role(s: Research & Development)
Image:T.jpg
Name: student
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:PCRgroup9.jpg
The Open PCR machine is a device that is used to duplicate DNA strands. The machine uses heating and cooling techniques to separate dual helix strands of DNA and replicate those strands, therefore generating new DNA. Primers, which are custom built, short pieces of DNA are placed inside the machine along with the DNA. These primers act as a landing pad for DNA polymerase, a complex and naturally-occurring protein that copies a cell's DNA then divides the double helix into two separate parts.The PCR machine completes cycles that consist of heating the existing DNA until the two strands separate, cooling the DNA so primers can attach onto the strand, and then heating up the DNA again so that DNA polymerase can copy and duplicate the strands After the PCR completes thirty to forty cycles, billions of new strands of DNA are created. .


Experimenting With the Connections

After unplugging part 3 from part 6 of the PCR machine, the LED screen shut off. Once we reconnected the wire, the light came on again. From this information we can conclude that parts 3 and 6 are connected to the bulb that lights up the screen.

When we unplugged the white wire that connects (part 6) to (part 2), the temperature on the screen dropped.


Test Run

The date that we first worked with Open PCR was October 22, 2013 at 9:00 am. The number of the machine our group was assigned was 10.




Protocols

Thermal Cycler Program


DNA Sample Set-up

Positive Control:
Cancer DNA template
Tube Lable: PC
Patient 1
ID:90616
Replicate 1
Tube Lable: 1R1
Patient 1
ID:90616
Replicate 2
Tube Lable: 1R2
Patient 1
ID:90616
Replicate 3
Tube Lable: 1R3
Negative Control:
Non-Cancer DNA template
Tube Lable: NC
Patient 2
ID: 91514
Replicate 1
Tube Lable: 2R1
Patient 2
ID: 91514
Replicate 2
Tube Lable: 2R2
Patient 2
ID: 91514
Replicate 3
Tube Lable: 2R3


DNA Sample Set-up Procedure

  1. Recieve Patient ID
  2. Clearly label each tube with the Patient and Replicate Number
  3. Using a disposible pipette, in each tube put 50μL of the PCR Reaction Mix. Do not reuse the pipettes, get a new one for each measurement.
  4. In each tube add 50μL of the DNA/primer mix into their respective tubes

PCR Reaction Mix
The PCR reaction mix contains taq DNA polymerase, MgCl2, and dNTP's. In each of the 8 tubes there will be 50μL of solution


DNA/ primer mix
The Dna/primer mix will contain the same forward and reverse primers in each solution, however each tube will contain a different template of DNA. Each tube will have 50μL of this mix.




Research and Development

PCR - The Underlying Technology

PCR has various components. The multiple aspects have an important role in multiplying and duplicating DNA. The PCR also has an important role in a sensitive test for tissue typing and vital for organ transplants but the main cause we will be studying is the study of patterns of gene expression. At the start of the process, a template DNA labels the DNA selected to copy and the RNA protein which will be created. Primers are the attaching component to the PCR reaction. Simultaneously, the primers copy the DNA and stop the polymerase from attaching to the template DNA. A Taq polymerase are high resisting heat molecules that attach to the primers to match the nucleotides and copy the DNA. Magnesium Chloride is the catalyst within this reaction which helps to tag the polymerase. The last component of a PCR reaction are deoxyribonucleotides abbreviated as (dNTP's) which pairs up with the complementary bases to replicate DNA strands. During thermal cycling the samples are heated up at ninety-five degrees Celsius for three minutes. Then at the same temperature for thirty seconds the DNA is denatured, where the helix separates creating two separate strands. The temperature then drops to fifty-seven degrees Celsius for thirty seconds where the primers anneal to their complementary strands, Adenine pairs with Thymine and Cytosine pairs with Guanine. The temperature is again increased where the tag polymerase attaches at the priming site and extends to form a brand new DNA strand. The last step stays at the same temperature where the polymerase continues to the end of the strand until it falls off. The final hold is dropped from seventy-two to four degrees Celsius to prevent potential nonspecific binding of primers and amplification.

Components of a PCR reaction. Magnesium Chloride is not pictured.


Image:PCRGroup9.png

(Source: www.extension.org/pages/32364/the-polymerase-chain-reaction-pcr#.UnBgFvmsidk)





Personal tools