BME103:W930 Group9

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: Tyler Ray Research and development scientist Name: Seth Howell Computer Genius Name: Ryan Open PCR machine engineer Name: Hamas Role(s) Name: Deanna Open PCR machine engineer LAB 1 WRITE-UP (Please finish by 11/7/2012) Initial Machine Testing The Original Design The open PCR is a device that enables the splitting of DNA. It is run through many cycles of heating and cooling that enables the splitting and recombination with polomers. It connects to a computer program to run the cycles. The temperature change is controled by the heating lid and than the information is fed to the LED screen. The heating lid heats tubes that are held in the heat tube holder to the appropriate temperature and then cools them as needed in each cycle. Experimenting With the Connections When we unplugged part (part 3) from (part 6), the machine ... (did what? fill in your answer) When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer) Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Polymerase Chain Reaction Polymerase Chain Reaction uses thermal cycling to artificially replicate DNA in vitro. First, a piece of double stranded DNA is added to a test tube containing a solution of primers, and the enzyme DNA polymerase. This solution is heated to 95 degrees celsius in order to separate (denature) the DNA molecule. Then, the solution is cooled to 57 degrees celsius and the primers bind to the single strands of DNA (provided the base sequences match up), then the solution is heated to 72 degrees celsius and DNA polymerase adds bases to complete the new complimentary strands, doubling the amount of DNA. This is repeated several times. The Master Mix GoTaq colorless Master Mix was used in this experiment. It contains taq polymerase, dNTPs(bases), MgCl2, and reaction buffers. Specifically, 2x colorless GoTaq master mix contains 400um of each base, and 3mm MgCl2 and a reaction buffer of pH 8.5. Part of this was then mixed with a few other ingredients as detailed in the table below. (information from www.promega.com) Contents of PCR vials Reagent Table Volume Template DNA (20 ng) .2 μL 10 μM forward primer 1.0 μL 10 μM reverse primer 1.0 μL GoTaq master mix 50.0 μL dH2O 47.8 μL Total Volume 100.0 μL Flourimeter Measurements (Add your work from Week 3, Part 2 here) Fluorimeter setup Instructions for opening images in imageJ 1. take a picture of the fluorimeter assembly with a smartphone. 2. transfer the picture to a laptop equipped with imageJ via icloud or email. 3. convert the file to a tiff. 4. click import in the imageJ options and select TIFF virtual stack. 5. find the image in the search box. and click on it. 6. The image is now open and can be analyzed. Research and Development Specific Cancer Marker Detection - The Underlying Technology (Add a write-up of the information discussed in Week 3's class) (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 OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.) Results (Your group will add the results of your Fluorimeter measurements from Week 4 here)