BME100 s2014:W Group5 L5

BME 100 Spring 2014	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: Garrett Sciascia Role: Mad Scientist Name: Juan Bahena Role: OpenPCR Technician Name: Blake Marx Role: WetWare Correspondent Name: Danielle Hallaran Role: Data Collection Name: Naomi Walliman Role: Team Manager LAB 5 WRITE-UP Background Information SYBR Green Dye A fluorescent DNA dye that binds all dsDNA and is measured by the change in fluorescence throughout. The dye is used in the PCR reaction in order to detect double stranded DNA. This allows experimenters to quantify the amount of DNA being produced. Single-Drop Fluorimeter An instrumentation that is able to detect fluorescence. The quantity is proportional to the number of molecules being deteced. How the Fluorescence Technique Works A technique that detects light in a molecule much similar to a spectrometer. It uses an ultraviolet light and excites the electrons causing the molecules to emit light. Procedure Smart Phone Camera Settings First Phone: Type of Smartphone: iPhone 5s Flash: Off ISO setting: 800 White Balance: Auto Exposure: Highest Setting Saturation: Highest Settings Contrast: Lowest Settings Additional Phone: Type of Smartphone: Galaxy Samsung S3 Flash: Off ISO setting: 800 White Balance: Auto Exposure: Highest Setting Saturation: Highest Setting Contrast: Lowest Setting Calibration First turn on the Blue LED switch in order to activate the excitation light. Also make sure the camera is open with the settings listed above. From there place the pone on the cradle with 90 degree angle from the slide. Make sure to adjust the height of phone and/or fluorimeter in order to take pictures of the drops. Lastly, the cradle should be as close to the fluorimeter as possible with having a clear picture still. Distance between the smart phone cradle and drop = 4.5 cm Solutions Used for Calibration Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Volume of the 2X DNA solution (μL) Volume of the SYBR GREEN I Dye solution (μL) Final DNA concentration in SYBR Green I solution (μg/mL) 5 80 80 2.5 2 80 80 1 1 80 80 0.5 0.5 80 80 0.25 0.25 80 80 0.125 0 80 80 0 Placing Samples onto the Fluorimeter [Instructions: Step one, in your OWN words] [Instructions: Step two, in your own words] [Instructions: Step three, in your own words] [Instructions: Step etc., in your own words] Data Analysis Representative Images of Samples [Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA] [Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with DNA (positive signal)] Image J Values for All Samples PCR Product TUBE LABEL Volume of the DILUTED PCR Product solution (µL) Volume of the SYBR GREEN I Dye solution (µL) INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS AVERAGE INTDENS VALUE A1 80 80 2716687 3229970 3340085 3095581 A2 80 80 1451878 2568844 8446955 4155892 A3 80 80 3862714 8011744 1600043 4491500 B1 80 80 9856858 5058003 3108259 6007707 B2 80 80 6862644 5685363 6783718 6443908 B3 80 80 6287959 7119245 9598254 7668486 + 80 80 11779932 7973600 11270538 10341357 - 80 80 4371681 2482949 1977785 2944138 Fitting a Straight Line [Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]