BME100 f2013:W1200 Group9 L4

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
OUR TEAM File:.jpg Name: Jessica Stradford Role(s): Open PCR machine testing File:.jpg Name: Rachael Hall Role(s): Open PCR machine testing File:.jpg Name: Kenna Lum Role(s): Protocol Planning File:.jpg Name: Michelle Sigona Role(s): Research & Development File:.jpg Name: Salvador Avina Role(s: Research & Development) File:T.jpg Name: student Role(s) LAB 1 WRITE-UP Initial Machine Testing The Original Design 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 Recieve Patient ID Clearly label each tube with the Patient and Replicate Number 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. 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. (Source: www.extension.org/pages/32364/the-polymerase-chain-reaction-pcr#.UnBgFvmsidk)