BME100 s2014:T Group10 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 Sean-Christopher Bradbury Thursday Group 10 Member Suyen Go Thursday Group 10 Member Nathaniel Kirkpatrick Thursday Group 10 Member Maya Robinson Thursday Group 10 Member Lissette Valle Thursday Group 10 Member LAB 5 WRITE-UP Background Information SYBR Green Dye SYBR Green Dye is a dye that stains nucleic acids by binding to DNA or RNA. The dye is used to visualize DNA through staining agarose gels, biochip applications, fluorescence imaging techniques, real-time PCR, and more. SYBR dye fluoresces when combined with dsDNA (Zipper Hubert Brunner 1). However, unbound SYBR Green is unreactive to light. This feature allows scientists to determine the concentration of a target DNA sample by measuring the intensity of the fluorescent light being emitted by the sample. The higher the intensity of the light being emitted, the more concentrated the sample is with the target DNA. SYBR Green, as its name implies, fluoresces with a green color. Accordingly, it is important to use a different wavelength of light to excite the sample in order to prevent distorting the results. There are several types of fluorescent dyes similar to the SYBR Green used in this lab, but SYBR Green was chosen for its environmental friendliness and high level of light emission intensity to allow for easier detection of binding to the target DNA. Single-Drop Fluorimeter A fluorimeter is a device used to measure and identify fluorescence in a medium. Fluorimeters measure certain wavelengths of light and have two light detectors, one of which detects absorbency while the other detects fluorescence emission (So Dong 1). Single-Drop Fluorimeters do this with a single drop of a sample that is placed on a hydrophobic slide in the path of a beam of single wavelength light. For example, in our setup we used blue LED light to allow for light absorption and prevent contaminated intensity data that would occur if we used a green LED since the SYBR Green Dye fluoresces in the green range. A light detector or camera placed at a uniform distance from this sample is then used to capture the intensity of the fluorescence being emitted from the sample. Cameras are acceptable for this laboratory set-up even though they are unable to measure light intensity because it is assumed that light intensity is proportional to brightness; the brighter an object is in a photo, the higher the light intensity. (Figure 1: Our Single-Drop Fluorimeter setup. A sample is placed in the path of the LED light on the white hydrophobic slide and its light intensity is captured by taking a picture with the iPhone. Image by Nathan Kirkpatrick) (Figure 2: Our Single-Drop Fluorimeter covered by a box to prevent ambient light interference. A timer is used to take the picture of the sample once the box is closed. Image by Nathan Kirkpatrick) How the Fluorescence Technique Works In this experiment, the concentration of DNA was determined by measuring the amount of fluorescence in a medium which had been dyed with SYBR Green Dye. Since light intensity is assumed to be proportional to brightness, images taken with a camera using the Single-Drop Fluorimeter setup described above can be used to determine the intensity of the SYBR Green Dye and, accordingly, the concentration of target DNA in the sample. After a single-drop fluorimeter was used to measure the fluorescence and an image was taken, ImageJ software was used to determine the concentration of DNA. Citations: Zipper, Hubert, Herwig Brunner, Jurgen Bernhagen, and Frank Vitzthum. "Investigations on DNA Intercalation and Surface Binding by SYBR Green I, Its Structure Determination and Methodological Implications." Ncbi.nih.gov. US National Library of Medicine, 12 July 2004. Web. 3 Apr. 2014. So, Peter TC, and Chen Y. Dong. "Fluorescence Spectrophotometry." Scribd. Macmillan Publishers Ltd, Nature Publishing Group, 16 Jan. 2009. Web. 03 Apr. 2014. Procedure Smart Phone Camera Settings Type of Smartphone: Apple iPhone5S Flash: no flash ISO setting: 2500 White Balance: Auto Exposure: Highest Setting Saturation: N/A Contrast: N/A Calibration [Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.] Distance between the smart phone cradle and drop = 8cm [Instructions: See worksheet page 6.] Solutions Used for Calibration [Instructions: See worksheet page 6.] row 1 cell 1 row 1 cell 2 row 1 cell 3 row 1 cell 4 row 2 cell 1 row 2 cell 2 row 2 cell 3 row 2 cell 4 row 3 cell 1 row 3 cell 2 row 3 cell 3 row 3 cell 4 [Add more rows as needed] 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 (Figure 3: ImageJ Green-Channel isolated Negative Control image with circle around droplet) (Figure 4: ImageJ Green-Channel isolated Positive Control image with circle around droplet) 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 PCR Product Tube Label Area (pixel^2) Mean Pixel Value RawIntDen of the Drop Average RawIntDen of the Drop PCR Product Concentration ug/mL Corrected PCR Product Concentration, ug/mL PC 1 30528 123.781 3778777 3651602.667 1.651602667 19.819232 PC 2 28794 124.966 3598262 PC 3 28349 126.204 3577769 NC 1 26035 49.289 1283252 1038869.667 -0.961130333 -11.533564 NC 2 25986 38.364 1002239 NC 3 24081 34.513 831118 1R1 1 26780 40.389 1081609 1088399 -0.911601 -10.939212 1R1 2 28678 38.826 1113459 1R1 3 27348 39.13 1070129 1R2 1 27328 43.119 1178362 1253375.333 -0.746624667 -8.959496 1R2 2 29178 45.662 1332320 1R2 3 26307 47.495 1249444 1R3 1 22214 44.459 987619 891381 -1.108619 -13.303428 1R3 2 26264 32.101 843096 1R3 3 25368 33.248 843428 2R1 1 22296 126.872 2828741 3107452.667 1.107452667 13.289432 2R1 2 25085 130.523 3274161 2R1 3 24628 130.723 3219456 2R2 1 26100 129.338 3375712 3274541 1.274541 15.294492 2R2 2 26272 128.394 3373160 2R2 3 23711 129.676 3074751 2R3 1 23900 128.988 3082802 3275054.667 1.275054667 15.300656 2R3 2 23824 131.695 3137491 2R3 3 27428 131.43 3604871 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 concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as 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 _____ : 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 concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as 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 _____ :