BME100 s2014:W Group3 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: Joseph Roskop Name: Lorelei Fyfe Name: Ana Kenia Viera Name: Ben Lenschow Name: Adnan Alsharif Name: Group Picture LAB 5 WRITE-UP Background Information SYBR Green Dye SYBR Green Dye is a nucleic acid stain used to identify isolated segments of DNA. Usually utilized to determine whether or not genetic pathogens or discrepancy's are present. Capable of detecting Virus's like HIV as well as precursors for Diabetes. Single-Drop Fluorimeter The Single-Drop Fluorimeter is the device that illuminates the dye allowing for both qualitative and quantitative analysis of the compounds being observed. The device itself consists of just a box stand with a slide holder on its surface. Lateral to the stand is a small fluorescent bulb that illuminates the fluid solution. On the side of the box is a flip switch that turns the fluorescent light on and off. How the Fluorescence Technique Works The Fluorescence Technique's function is to identify the presence of different genetic pathogens. This works by bathing the SYBR Green Dye and DNA sample with a black light. A phenotype present in target DNA sequences causes positively effected DNA samples to glow with a green fluorescence when subjected to the black light. Procedure Smart Phone Camera Settings Type of Smartphone: Samsung Galaxy S4 Flash: Inactive ISO setting: Auto White Balance: Auto Exposure: Highest Setting Saturation: Highest Setting Contrast: Lowest Setting Calibration The smart-phone is set in a position so that its camera is eye level with the slide. In order to achieve this with phones of different lengths, it is necessary to raise the box off the ground using small booklets or pencil boxes. For the most effective images the phone timer is set so that the image is taken after the lid of the blackout box is closed. Distance between the smart phone cradle and drop = 14cm Solutions Used for Calibration Initial Concentration (micrograms) Volume of the 2x DNA solution Volume of SYBR GREEN 1 FInal DNA concentration in SYBR GREEN 1 solution 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 a slide with the smooth side back down in the cut out slot in the flourimeter. Place the cellphone on the cradle with the camera facing the flourimeter. Adjust the flourimeter to the necessary height using booklets or trays. Measure the distance between the cellphone and the flourimeter. Put a 80 microliter drop of SYBR Green 1 on the slide on the flourimeter directly across from the fluorescent light bulb. Add another 80 microliter drop of your sample on the top of the drop already on the slide. Make sure that the light is switched "ON" Using the cellphone and timer press the capture button on the phone. Put down the lid of the light box. Allow phone to take the picture and then lift the lid. Record what sample you just took a picture of and repeat this with the next position. 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)] PCR Results Summary Eight PCR reactions were completed in previous lab. This lab used the SYBR Green I technique to clearly see the amount of amplified DNA in each reaction. Positive control : PCR result 2,428,578 μg/mL Negative control : PCR result 935,166 μg/mL Patient 80926 : A basic clear shiny drop : Recordings were 325,798 μg/mL, 259,300 μg/mL, and 734,163μg/mL. These numbers are smaller compared to the positive control value and closer in comparison to the negative control. This concludes that the first patient had a negative test result. Patient 83588 : A bright green fluid drop : Recordings were 3,717,326 μg/mL, 3,488,620 μg/mL, and 2,897,463 μg/mL. These numbers are larger in comparison to the negative control value and closer in comparison to the positive control value. This concludes that the second patient had a positive test result. Image J Values for All Samples Final DNA Concentration in SYBR Green I Solution (μg/mL) RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDENdrop - RAWINTDENbackground 5 10405213 2813 10402400 2 9621172 10633 9610539 1 3329855 27440 3302415 0.5 992867 4311 988556 0.25 1176715 17555 1159160 0 2203865 3767 2200098 Negative1 (0.5) 660226 4104 656122 Negative2 (0.5) 3572442 21428 3551014 Negative3 (0.5) 1198328 2781 1195547 Positive1 (0.5) 5181375 17736 5163639 Positive2 (0.5) 4542836 8532 4534304 Positive3 (0.5) 4847254 6815 4840439 A1 (0.5) 643172 4177 638995 A2 (0.5) 742985 7540 735445 A3 (0.5) 568579 3477 565102 B1 (0.5) 647707 3540 644167 B2 (0.5) 485750 2628 483122 B3 (0.5) 422343 6012 416331 C1 (0.5) 1123599 4395 1119204 C2 (0.5) 2561727 4104 2557623 C3 (0.5) 719651 17872 701779 X1 (0.5) 4696847 5220 4691627 X2 (0.5) 8815318 6399 8808919 X3 (0.5) 8791792 17908 8773884 Y1 (0.5) 7017068 13448 7003620 Y2 (0.5) 4720939 8402 4712537 Y3 (0.5) 9193710 4547 9189163 Z1 (0.5) 6168229 7129 6161100 Z2 (0.5) 5532864 7209 5525655 Z3 (0.5) 5683683 7794 5675889 Fitting a Straight Line Image:BME100Graph.jpg