BME103 s2013:T900 Group6

BME 103 Fall 2012	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Cyril Wassef -Initial Machine Tester Name: Israel Brewer Research and Development Name: Dale Franco L. Caagbay - Protocol Role(s) Name: Emmanuel "Manny" Casildo Research and Development Name: student Role(s) Name: student Role(s) LAB 1 WRITE-UP Initial Machine Testing The Original Design Description: This is a PCR machine. It is a piece of equipment that allows scientists to create different amounts of DNA sequences. With it, scientists are able to denature (break down) specific DNA strands by inducing heat, and thus are able to analyze them specifically. This machine helps with research on possible DNA mutation, and aids in on-going research to help resolve today's problems with diseases such as cancer. Experimenting With the Connections When we unplugged the LCD screen from the OpenPCR circuit board, the machine's LED failed to work properly. When we unplugged the white wire that connects the OpenPCR circuit board to the main heating block, the machine began to show a change of the temperature on the LCD screen. Test Run The date the PCR machine was open to conduct an analysis was on March 5th, 2013. We had machine number 6 that day. What we came to see, in terms of pros and cons, contained the following: Pros: - Lightweight - Quiet - Ease of Use Cons: - Took a while to complete the full cycle - The lid is not easy to remove. Requires some work - Requires a computer - Small and somewhat heavy - Made of Wood (fire hazard) Protocols Thermal Cycler Program DNA Sample Set-up Positive Control - Cancer Patient 1- Vial 1 Patient 1- Vial 2 Patient 1- Vial 3 Negative Control - No Cancer Patient 2- Vial 1 Patient 2- Vial 2 Patient 2- Vial 3 DNA Sample Set-up Procedure 1. Obtain DNA samples, PCR, DNA/Primer vials 2. Open All The Tops 3. Use pipette to move DNA into vials containing PCR mix PCR Reaction Mix What is in the PCR reaction mix? The PCR mix is made up of 50 Microliters of PCR. DNA/ primer mix What is in the DNA/ primer mix? The DNA/Primer Mix is made of 50 micro liters of DNA of patients 1 and 2, and primer. There are 2 vials containing negative and positive control. The negative control does not have cancer DNA, and the positive control has cancer in it. Research and Development How PCR Replicates DNA What is DNA? DNA, short for deoxyribonucleic acid, is located within the nucleas of a cell. DNA is shaped as a double helix with two strands on each side, some refer to it as a winding staircase. In between the strands there are things called nucleotides, or the stairs, which hold the DNA together. There are four different kinds of nucleotides that form base pairs when they are bonded together. These base pairs are Guanine-Adenine and Thymine-Cytosine. The Process The PCR is needed in the replication of DNA based on the fact that the PCR machine has the ability to heat and cool down the substances that are placed inside of it. These temperature changes are crucial in the replication process because at high temperatures DNA strands seperate into single strands. Before the tube is even placed in the PCR machine you will need more then just DNA inside the tube. Primers is the first thing to interact with the DNA. While the DNA is separated into two strands, primers, which are pretty much short single strands of DNA, bind to the DNA instead of letting the DNA bind together to itself again. This is where the nucleotide base pairs come in, each primer has the exact nucleotide sequence for the DNA in order to bind to it. The primers are to short to finish the whole DNA replication, polymerase comes in to finish the job with the help of magnesium chloride and dNTP, which just help grab nucleotides for the polymerase. The replication process happens over and over again due to the temperature control of the PCR machine until you have enough DNA to be observed. Bonus Once again, in our experiment, we used the PCR machine, also known as the polymerase chain reaction machine. It is used to amplify strands of DNA and generate many copies of a specific sequence. As previously stated, it can be used to research different diseases, especially cancer. When it comes to replicating DNA, the PCR machine requires, of course, a DNA template strand and a primer to start off the replication process. Also needed in the process is DNA Polymerase which adds base pairs to the new strand of replicated DNA (the daughter strand, in other words). In the case of a cancer DNA template, when it comes time to replicate, a primer sets down base pairs for DNA Polymerase to have a basis to start, and leaves until DNA Polymerase is finished adding new base pairs. For this process, a “Taq” Polymerase is named for the protein that assembles these base pairs on the new DNA template strand. In addition, for Taq Polymerase to function, magnesium chloride must be present. This all can happen only if a cancer gene coded in the sequences of the DNA strand is present. If there is no cancer gene, cancer DNA replication will not happen. As we can see in this illustration, this is how the PCR Machine functions. In order to create copies of the DNA strands it's given, the DNA is put through a series of temperature changes. Over a period of time, this allows the DNA strand being copied to unravel, grow new strands, or lock in new strands on the template. In this comparison, the polymerase on the left allows the viewer to see the DNA template brighter and more accurately. Depending on what amplification you use, results can either come out being better to see and compare or difficult to see and make accurate observations on exactly what is the result. Sources (http://www.ornl.gov/sci/techresources/Human_Genome/publicat/primer/pcr.html). (http://dangerousprototypes.com/wp-content/media/2011/07/OpenPCR_solo-W490.html). (http://media.treehugger.com/assets/images/2011/10/open-pcr-inside.html). (http://www.scilproj.org/DNA%20amplification%20by%20PCR.html). (http://www.invitrogen.com/etc/medialib/en/images/ics_organized/applications/nucleic_acid_amplification/data_chart.html).