Name: Ryelle Pattuinan
Research and Development
Name: Kenze Caulfield
Name: Jackie Janssen
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
This is an Open PCR machine. The purpose of the machines is to fluctuate the temperature of different samples of DNA strands to separate them into a single strand. It allows for a way to amplify DNA sequences by this separation and heating them with primers and DNA polymerase. When raised to a high enough temperature DNA melts, which is where it separates into the two strands, and primers and DNA polymerase are used to fill in the holes and attach to the now single-stranded DNA. The machine is able to amplify a specific sequence of DNA up to 1 billion times. Because of this ability, scientists and investigators can use it to amplify these sequences even if only a small amount of DNA is provided. Single roots in hair or even microscopic splatters of blood left such as at scenes of crime are actually ample amounts of DNA for PCR.
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine's display screen didn't turn on or show up.
When we unplugged the white wire that connects (part 6) to (part 2), the machine's temperature reading was altered compared to the accurate values from the computer.
We first tested Open PCR on October 25, 2012. The machine was accurate and precise but the cycling was slow.
Polymerase Chain Reaction
The Polymerase Chain Reaction works by attaching MgCl to taq enzyme in the solution and then pulling deoxy nucleotide diphosphates, binding them to the strand that is already present. This gives a base to the DNA that is already there.
DNA amplification contains three important steps: heat denauturation,primer annealing, and primer extension.
Step One: Heat Denauturation-Heat is applied to the DNA and the two strands separate.
Step Two:Primer Annealing-With excess dNTPs, oligonucleotides are added.
Step three: Primer Extension-DNA polymerase is added and new strands of DNA are synthesized. The DNA strands combine with the nucleotides to form completed, complimentary strands of DNA.
PCR master mix contains Magnesium Chloride, all four Nucleotides needed to create DNA, and reaction buffers.
Template DNA(20 ng) .2uL
10 uM forward primer 1.0 uL
10 uM reverse primer 1.0 uL
GoTaq master mix 50.0 uL
DH2O 47.8 uL
Total Volume 100.0 uL
Patient one's ID number is 12123, male, and 61 years old. Patient two's ID is 21312, female, and 56 years old.
The Flourimeter was assembled by taking the black box, flipping it upside down so that the rest of the materials can be placed inside the box with the flap blocking out excess light. The glass slide was then placed into the flourimeter so that the light of the flourimeter was on the first two rows of holes in the glass slide. The flourimeter was then placed inside the overturned black box, and the phone was placed on the stand. The camera on the phone is lined up with the middle row of holes in the glass slide. After the pictures of the droplets of DNA and SYBR green solution were taken, they were sent to a computer through e-mail and downloaded.
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
(Add a write-up of the information discussed in Week 3's class)
(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 OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)
| Sample || Integrated Density || DNA μg/mL || Conclusion
| PCR: Negative Control || E6 || F6 || G6
| PCR: Positive Control || E7 || F7 || G7
| PCR: Patient 1 ID 12123, rep 1 || E8 || F8 || G8
| PCR: Patient 1 ID 12123, rep 2 || E9 || F9 || G9
| PCR: Patient 1 ID 12123, rep 3 || E10 || F10 || G10
| PCR: Patient 2 ID 21312, rep 1 || E11 || F11 || G11
| PCR: Patient 2 ID 21312, rep 2 || E12 || F12 || G12
| PCR: Patient 2 ID 21312, rep 3 || E13 || F13 || G13
- Sample =
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