BME100 s2014:W Group11 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: Christopher Stark Name: Kandace Donaldson Name: Cassiana Wright Name: Jordan Nelson Name: Yash Jain LAB 5 WRITE-UP Background Information SYBR Green Dye SYBR Green I is a fluorescent dye used for binding double-stranded DNA so that quantification and visualization is possible. SYBR Green is a synthetic, asymmetrical cyanine dye that was designed to work throughout a wide range of temperatures, bind in a way that does not interfere with enzymes such as DNA polymerases and nucleases, and accurately quantify DNA in varying concentrations. Real time PCR is the most common use for the dye, where the fluorescence increases as each PCR cycle amplifies the DNA so you can visually observe amplification. Single-Drop Fluorimeter The Single-Drop Fluorimeter is a device designed to help detect any presence of double stranded DNA in a liquid sample by measuring its fluorescence. It is essentially a small plastic device with a space on top for a multi-welled slide. The slide is illuminated by a single blue LED light which is turned on by a switch located on the right hand side of the Fluorimeter. The slide of the fluorometer is positioned so that the blue LED can iluminate any drop placed over a designated well. [Instructions: A description of the single-drop fluorimeter device. Add a PHOTO for bonus points] How the Fluorescence Technique Works The fluorescence technique works by adding a fluorescent die to a liquid with an unknown percentage of double stranded DNA. The slides used in this analysis have a hydrophobic Teflon film on one side of the slide, designed to hold each sample in place. The film contains breaks which act as wells where liquids can be held in precise quantities. These breaks also keep each of the drops apart from one another so that the samples do not mix. All these traits make it easier to observe and analyze our samples when using the LED. When the samples are placed in the LED's range, any DNA stands at the top of the drop sized sample can be seen because the light causes them to fluoresce. Procedure Smart Phone Camera Settings Type of Smartphone: Nokia Lumia 928 Flash: Off ISO setting: 3200 White Balance: Auto Exposure: 2 Saturation: N/A Contrast: N/A Calibration The camera was placed in a cradle that kept it upright and facing the fluorimeter from a side angle nearly edge-on. It was kept at a distance of 13 centimeters from cradle to the drop, which was chosen to keep the drop in focus for the camera and remained constant throughout the experiment. An eraser was placed in the cradle with the phone in order to keep it snug against the side and in a consistent position. It was also necessary for the fluorimeter to be raised to a height that was suitable for the height of the camera so it was placed on top of a calculator. Distance between the smart phone cradle and drop = 13 centimeters Solutions Used for Calibration [Instructions: See worksheet page 6.] Initial Concentration of 2X Calf Thymus DNA Solution (μg/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 [Add more rows as needed] Placing Samples onto the Fluorimeter Place the slide, smooth side down, in the fluorimeter (make sure it's on). Check the height of the flourimeter and camera position. Pipet 80μL drop of the SYBR Green I solution between the first two clear circles in the middle of the slide. Pipet 80μL drop of the sample solution on top of the SYBR Green I drop. Move the slide so that the fluorimeter's light illuminates the center of the drop and focuses light on the other side. Place the lightbox over the setup with the flap still open and check the camera focus again. Take picture with the camera using a set timer with the lightbox flap down. Check the picture. Pipet the drop off of the slide and into the liquid waste container. Move the slide to the next set of clear circles in preparation for the next drop. Repeat steps until all slide positions have been used and then replace the slide with a new one. 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 [Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.] TABLE GOES HERE Fitting a Straight Line [Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9] PCR Results Summary Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concnetration of claf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples are used to determine the threshold values for determining whether an unknown (patient) sample is truly positive or negative. Your positive control PCR result was ____ μg/mL Your negative control PCR result was ____ μg/mL Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed Patient _____ : Patient _____ : Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion. Patient _____ : Patient _____ :