BME100 s2014:W Group12 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: RJ Parkinson Name: Jacqueline Stokes Name: Austin Doyle Name: Anthony Zlatket Name: Sara Jerez LAB 5 WRITE-UP Background Information SYBR Green Dye The SYBER I Green dye is used in this lab to visually detect amplified samples of DNA. SYBER green acts as a dye molecule that emits a florescent green color in the presence of double stranded DNA. To detect this light the sample is mixed with SYBER green and placed under a black box to block out interfering surrounding light. Then using a smartphone to take an image of the mixture the we can analyze the brightness of the dye and how much of the dye fluoresces to indicate the amount of DNA in the sample. Single-Drop Fluorimeter This lab uses a single drop fluoremeter device to detect DNA samples. This device consists of a sturdy outer cover which serves to block out interfering light sources so that the fluorescent light is more easily detected. We set up a phone cradle to secure the phone while it is taking pictures. Inside the device there is a platform for the slides to be placed on a surface designed to repel water so droplets on the slide maintain a spherical shape. Around the slide there is a LED light source which emits a blue light in order to clearly see the fluorescing dye. How the Fluorescence Technique Works The blue LED light shines on the sample underneath the black box, in the dark, and detects the different DNA concentrations. The samples are shown to have different DNA concentrations based on the amount of light that they fluoresce. Procedure Smart Phone Camera Settings Type of Smartphone: iPhone 5C Flash: None ISO setting: Auto White Balance: Auto Exposure: Auto Saturation: Auto Contrast: Auto Did not arrange the settings on the phone. But the lab manual says that this is acceptable because the camera is most likely sensitive enough. Calibration The phone is placed upright in the cradle with an eraser to help it stand straight. The cradle is moved about 6.5 cm away from the fluorimeter. This set up never moves once the experiment has begun. Distance between the smart phone cradle and drop = 6.5 cm Solutions Used for Calibration 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 Step 1: Insert the slide, smooth side down Step 2: 80 milliliters of the SYBR GREEN on the first slide position Step 3: 80 milliliters of the calf thymus (water blank) on top of the "beach ball" of SYBR GREEN Step 4: Focus camera on the "beach ball", cover the fluorimeter with the black box, and take a picture in the dark Step 5: Repeat this process on different slide placements (at least three) for each sample. Data Analysis Representative Images of Samples Image with No DNA Image with DNA Image J Values for All Samples PCR Product Tube Label Diluted PCR Product solution volume (µL) SYBR Green 1 Dye solution volume (µL) INTDENS values INTDENS values INTDENS values Average INTDENS value RJ1 80 80 925556 370350 NA 647953 RJ2 80 80 80765 52661 30813 54746 RJ3 80 80 47062 39069 51594 45908 RJ4 80 80 54742 31713 58904 48786 RJ5 80 80 50946 56850 51390 53062 RJ6 80 80 58585 57683 65830 60699 RJ7 80 80 54772 55469 97643 59294 RJ8 80 80 56383 54353 57477 56071 Fitting a Straight Line PCR Results Summary Our positive control PCR result was 30.20 μg/mL. Our negative control PCR result was -36.08 μg/mL Patient 78086 : The images of the three samples of DNA given from Patient 78086 were without color. There was no sign of any SYBR Green 1 in all three images. The amount of corrected PCR product concentration was around -36 μg/mL. The exact values were: -35.89, -36.88 and -36.56 μg/mL. Patient 32445 : The images of the three samples of DNA given from Patient 32445 were without color as well. Again there was no sign of any SYBR Green 1 in all three images. The amount of corrected PCR product concentration was around -35 μg/mL. The exact values were: -35.23, -35.38 and -35.74 μg/mL. Patient 78086 : Negative. We were able to come to this conclusion based on the values of μg/mL for both the positive and negative controls. This patient's corrected PCR product concentration matched that of the negative control. Patient 32445 : Negative. We were able to come to this conclusion based on the values of μg/mL for both the positive and negative controls. This patient's corrected PCR product concentration matched that of the negative control as well. We did not get confused on the results and assume that the results of the patients would be different. They in fact were very similar and we were able to successfully ascertain the desired result.