BME100 s2014:T Group3 L4

BME 100 Spring 2014	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
Kazhan Kader, Jaquelyn Corr, Sayer Aldaady, Hannah Spehar, Maria Morrow Name: Maria Morrow Name: Kazhan Kader Name: Hannah Spehar Name: Sayer Aldaady Name: Jaquelyn Corr Name: Group 3 LAB 1 WRITE-UP Initial Machine Testing The Original Design The OpenPCR is a DIY machine that separates DNA strands through heat. Once the strands are separated, one can read a DNA sequence and look for mutations or abnormalities. The machine sends a current through the DNA to send the sequence to the CPU. The CPU then translates it onto the display where it shows a copy of the DNA. It can be used to diagnose diseases. The OpenPCR copies the DNA from genotypic language to computer language then to high level language. Experimenting With the Connections Part 1- Heated Lid This part of the machine heats the DNA, subsequently denaturing it. Without this piece, the DNA would not separate. Part 2- 16 Tube PCR Plate The DNA is placed in this part and held while the machine run. Part 3- Output Screen The output screen displays the copy of the DNA and the status of the process. Part 4- Heater and Fan Without this, the DNA could not heat and the fan could not run to cool the rest of the parts of the machine down. Part 5- The CPU This is the central processing unit. It is what runs the machine and sends the data output to the display and to the connected computer. Part 6- Circuit Board Without the circuit board, there would be no current to be run through the DNA and there would be no information to read. When we unplugged (part 3) from (part 6), the display did not turn on. When we unplugged the white wire that connects (part 6) to (part 2), the heater and fan did not function. Test Run The date the OpenPCR was first tested was on March 20, 2014. The lid was first heated to 100°C. Then it was cooled to 95°C for 2 minutes and ran for 35 cycles. The DNA denatured at 95°C for 30 seconds. The annealing process cooled the DNA to 57°C for 30 seconds. It was then extended at 72°C for 30 seconds. Finally, the DNA was held at 72°C for 2 minutes, and it's final hold temperature was 4°C. The run was then started and finished successfully. Protocols Thermal Cycler Program DNA Sample Set-up Positive Control: (+) Patient 1, Replicate 1: (541) Patient 1, Replicate 2: (542) Patient 1, Replicate 3: (543) Negative Control: (-) Patient 2, Replicate 1: (431) Patient 2, Replicate 2: (432) Patient 2, Replicate 3: (433) Patient 1 Identification number: 54174 Patient 2 Identification number: 43184 DNA Sample Set-up Procedure Label 2 groups of 4 attached PCR tubes as illustrated in the previous table and place in rack. Mix PCR reaction mix (50uL) and Template DNA + primer mix (50uL) in 8 attached PCR tubes (labeled as previous table illustrates) Close lids tightly and place tubes in PCR machine Connect PCR machine to computer and open the OpenPCR application Run PCR machine PCR Reaction Mix The PCR Reaction Mix consists of Taq DNA polymerase, magnesium chloride, and dNTPs. DNA/ primer mix The DNA/primer mix cosists of the patients' DNA samples and the forward and reverse primers. Research and Development PCR - The Underlying Technology The component of template DNA is the DNA that is going to be amplified in the PCR reaction. Primers are stands that are complementary to the 3' template DNA strand used to be replicated. Taq polymerase is a DNA polymerase helps in DNA replication to synthesize DNA. The dNTP's are the building blocks from which the DNA polymerase synthesizes new DNA. The initial step of 95 C for 3 minutes activates the DNA polymerase. The denature step at 95C for 30 seconds causes the DNA to melt breaking the hydrogen bonds that hold the two strands together. The anneal step at 57C for 30 seconds allows primers to anneal to strands of DNA, starting the process of forming the DNA. The extend step at 72C for 30 seconds allows complementary strand of DNA to be synthesized using the dNTP's, exponentially synthesizing the DNA. Final step 72C for 3 minutes ensures that the strands of DNA keeps extending. The final hold 4C is for the storage of the DNA so no changes occur. The base pairs are as follows: Adenine (A) to Thymine (T) and Cytosine (C) to Guanine (G).