BME100 f2013:W1200 Group17 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: Sonia Malek Name: Micke Cox Name: Jason Yang Name: Kevin Loung Name: Andrew Prindle LAB 1 WRITE-UP Initial Machine Testing The Original Design br> PCR (short for polymerase chain reaction) is a technique used to multiply DNA molecules. Primers (short pieces of DNA made in the laboratory)and DNA polymerase (proteins that copy a cell's DNA before it divides in two) are two components that aid the PCR process by enabling the selected DNA to be replicated effectively. Both are inserted into the DNA in the PCR machine. Through a series of processes a PCR machine heats and cools the DNA that is inserted into the machine, replicating the DNA through a chain reaction. Experimenting With the Connections When we unplugged the LCD display (part 3) from the circuit board (part 6), the screen shut off. When we unplugged the white wire that connects the circuit board (part 6) to the heating plate (part 2), the recorded temperature was removed from the LCD display. Test Run Open PCR was first tested October 23, 2013. The machine was fairly cheaply made and the DNA replications were slow. The DNA replications did not happen at the rate expected.The PCR was slow and did not calibrate correctly when it was connected to the computer. The total time that was expected to run the heating and cooling protocol was 2 hours or less. Within 15 minutes during the operation of the machine, the test run was failing. The displayed results on the screen was slower than the expected compared to the computer monitor. As of result, the machine failed to pass the test run. Protocols Thermal Cycler Program DNA Set-up DNA Set-up Procedure Step 1) Label tubes according to the chart above. Of the 6 tubes given, 3 tubes must be for the Positive Control (Cancer DNA template) and 3 other tubes must be for the Negative Control (Non-Cancer DNA template). The ones that are for the Positive Control must be labeled #1P, #2P, #3P respectively. The tubes for the Negative Control must be labeled #1N, #2N, and #3N respectively. They can be labeled with a dry erase marker or with sharpie on a small piece of tape that is attached to each tube. Step 2) Move DNA/Primer mix into correct PCR tubes with a pipette tip (50 microliter in each). Only use each pipette tip once. Dispose of pipette tip after each transfer. Step 3) Put PCR reaction mix (50 microliter) into each PCR tube with pipette tip (reaction mix includes DNA polymerase, magnesium chloride, and dNTP's). Only use each pipette tip once. Dispose of pipette tip after each transfer. Step 4) Place PCR tube filled with all reaction components into a DNA Thermal Cycler (PCR machine) Step 5) Set the Open PCR Software to run a specific program in order to heat/cool the DNA appropriately. Make sure the HEATED LID portion is set at 100 degrees Celsius and the INITIAL STEP section is set at 95 degrees Celsius for 3 minutes. Make sure the DNA Thermal Cycler runs for 35 cycles denature at 95 degrees Celsius for 30 seconds, anneal at 57 degrees Celsius for 30 seconds, extend at 72 degrees Celsius for 30 seconds. Next it should be set at 72 degrees Celsius for 3 minutes, and the final hold should be at 4 degrees Celsius. Step 6) Close lid of DNA cycler and press start to collect data. PCR Reaction Mix The PCR reaction mix includes Tag DNA polymerase, MgCl2, and dNTP's. We used 8 tubes and each had 5 microliters of the mix in each. DNA/ primer mix Each DNA/Primer mix contains a different template DNA. All tubes have the same forward primer and reverse primer. 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.)