BME103:T130 Group 3 l2

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: Serena Kaplan Research and Development Name: Gabe McInnis Open PCR Machine Engineer Name: Blake Eichler Experimental Protocol Planner Name: Sierra Morris Experimental Protocol Planner Name: Zazu Moloi Open PCR Machine Engineer Name: Katelin Vaughn Research and Development LAB 2 WRITE-UP Thermal Cycler Engineering Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski. System Design Key Features Instructions Protocols Polymerase Chain Reaction PCR is a method of amplifying a sample of DNA. PCR alters the temperature and allows the DNA to separate, bind to primers, and catalyze. This results in the amount of DNA doubling after each cycle. Procedure: 1.) The DNA samples were heated to ninety-five degrees Celsius (95°C) for one (1) minute to unzip the two single strands. 2.) They were then cooled to fifty-seven degrees Celsius (57°C) and the primers were attached to their matching sequences. 3.) They were then heated back to seventy-two degrees Celsius (72°C) and polymerase extended the DNA strands by attaching the correct free nucleotides in order on the single strands. GoTaq Mix Components For 100μl reaction volume: 2X GoTaq Colorless Master Mix 10 μM upstream primer 10 μM downstream primer DNA template Nuclease-Free Water to Reagent Volume Template DNA (20 ng) 0.1μL 10μM forward primer 0.5μL 10μM reverse primer 0.5μL GoTaq master mix 25.0μL dH2O 23.9μL Total Volume 50.0μL Patient 1 ID 30269 Male, 55 years old Patient 2 ID 22057 Female, 55 years old Fluorimeter Setup 1.) The lid was first taken off the the box and one of its sides was unbuttoned in order to create a flap. 2.) The box was the flipped upside down in order to create a dark environment for the camera. 3.) A hydrophobic slide was then inserted into the flourimeter. 4.) Finally, the camera phone was placed in the stand. Fluorimeter Measurements Fluorimeter Assembly Procedure 1.) Label transfer pipettes and tubes 2.) Transfer each sample separately into tube containing 400μl of buffer 3.) Take the specifically labeled tube containing SYBR GREEN 1 and place 2 drops on the first 2 centered drops 4.) Place 2 drops of diluted sample on top of the SYBR GREEN 1 drop 5.) Align light through drop 6.) Take pictures using light box 7.) Repeat for each sample. 8.) Run water as BLANK using same procedure ImageJ Instructions 1.) Open ImageJ 2.) Click ANALYZE tool bar and select SET MEASUREMENTS 3.) Select AREA, MEAN GREY VALUE, and INTEGRATED DENSITY 4.) Upload image to ImageJ 5.) Select IMAGE then COLOR and then SPLIT CHANNELS 6.) Only use green channel 7.) Use OVAL tool and select the entire drop of liquid 8.) Go to ANALYZE and then MEASURE 9.) Drag circle to the background of the image 10.) Record results 11.) Repeat if necessary Research and Development Background on Disease Markers Primer Design Illustration