BME100 f2013:W1200 Group7 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 Name: Carlee Farhar Open PCR Machine Testing Name: Ambar Khare Open PCR Machine Testing Name: student Role(s) Name: student Role(s) Name: student Role(s) Name: student Role(s) LAB 1 WRITE-UP Initial Machine Testing The Original Design What is a PCR? A PCR machine stands for Polymerase Chain Reaction machine. When you get the PCR machine, you have to connect it to a computer via USB cable and have the OpenPCR software downloaded. This lets you in control of programming the PCR in a way you want it to work. The main function of this machine is that it uses a segment of DNA to generate billions of copies of that specific segment. DNA replication occurs naturally in the body where an enzyme known as Polymerase uses only one strand of DNA to make a complimentary strand which results in two double identical strands. A PCR however does this process repeatedly and within hours, it creates billions of copies in 35 cycles. The way it works is that the PCR goes through multiple heating cycles to produce duplicates of the segment of DNA. The very first step a PCR goes through is the process of heating up for three minutes. This is where the temperature rises up to 95 degrees C. After that, the denaturing step follows for 30 seconds which melts the DNA strand and splits the two strands apart connected by the sugar backbones. Second step is annealing for 30 seconds. This is where the temperature cools down to about 57 degrees C which then lets primers attach to the opposite ends of each strands of DNA (3' end). Because of the primers attached to the specific region of DNA, an enzyme called TAQ polymerase knows where to direct the synthesis of strands. This is known as the extending process which also lasts 30 seconds. TAQ polymerase gathers free nucleotides and binds them on each strand of DNA. The first cycle then comes to an end. The second cycle starts right away and instead of ending up with two strands, we end up with 4 strands. The next cycle we end up with 8 strands. The process keeps going on like this and we get our specific DNA segment to grow exponetially in hours. PCR Design (using third image above). There are several parts to the PCR design. Every part is dependent on each other. First there is the power source (heater) where it controls the temperature of the heating lid. The heating lid encloses the sample holder which holds the DNA segments you want to amplify. The fan is on the right side of the PCR which makes sure that the PCR doesn't overheat. The temperature rises up to 95 degrees C during the initial heating period and to cool it down, the fan turns on so overheating doesnt cause the whole machine to be shut down. Right underneath that is the circuit board which is the main component because it relays information to the rest of the PCR machine. When we open the PCR software on computer, we can choose what temperatures to keep the PCR in, time for each process, and the number of cycles and the circuit board relays this information to the rest of the parts. Finally, there is the LCD screen (blue) which displays information on what cycle the PCR is on and the temperatures. Experimenting With the Connections When we unplugged the LCD display (part 3) from the circuit board (part 6), the LCD display on the machine turned off. Part 6 was the circuit board and part 3 was the LCD screen display. The circuit board is the motherboard of the PCR machine. If the wire from that is disconnected from the LCD display sceen, then the circuit board wouldn't be able to relay information to the LCD screen on what needs to be displayed. When we unplugged the white wire that connects the circuit board (part 6) to the heating plate (part 2), the temperature dropped down. Test Run We started the test run of our PCR number 17 on Wednesday October 23rd at 12:55 p.m. After the test run, we concluded that the test run was a success and it ran exactly the way we programmed it on the software. Overall, we completed 25 of the 35 cycles which was enough to let us know if the device was working properly. Protocols Thermal Cycler Program DNA Sample Set-up row 1 cell 1 row 1 cell 2 row 1 cell 3 row 1 cell 4 row 2 cell 1 row 2 cell 2 row 2 cell 3 row 2 cell 4 DNA Sample Set-up Procedure Step 1 Step 2 Step 3... PCR Reaction Mix What is in the PCR reaction mix? DNA/ primer mix What is in the DNA/ primer mix? Research and Development PCR - The Underlying Technology (Add a write-up, essay-style, organized into paragrpahs with descriptive headers, based on the Q&A's from Section three of your worksheet) (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 PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)