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 Materials Supplied in the Kit PCR Machine 10μM forward primer 10μM reverse primer GoTaq master mix Supplied by the User Template DNA Distilled Water Pipets Eppendorf Tube PCR Protocol 1.) Mix 0.1μL of template DNA, 0.5μL of 10μM forward primer, 0.5μL of 10μM reverse primer, 25.0μL of GoTaq Master Mix, and 23.9μL of dH20. All of these items mixed together should create a total volume of 50.0μL. 2.) Up to 16 of these samples are then loaded into the PCR machine 3.) The DNA samples are then heated to ninety-five degrees Celsius (95°C) for one (1) minute in order to unzip the two single strands. 4.) They are then cooled to fifty-seven degrees Celsius (57°C) for ten (10) seconds so that the primers can attach to their matching sequences. 5.) Finally they are heated back to seventy-two degrees Celsius (72°C) for ten (10) seconds and polymerase extends the DNA strands by attaching the correct free nucleotides in order on the single strands. DNA Measurement Protocol 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 fluorimeter. 4.) Finally, the camera phone was placed in the stand. Fluorimeter Measurements 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