BME100 f2013:W1200 Group5 L5

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OUR TEAM Error creating thumbnail: File missing Name: Dominick Cocciola Error creating thumbnail: File missing Name: Hany Arafa Error creating thumbnail: File missing Name: Wendy Gray Error creating thumbnail: File missing Name: Kristina Roscher Name: Estefania Meza LAB 5 WRITE-UP Background Information SYBR Green Dye SYBR Green dye is a small molecular dye that fluoresces in the presence of DNA but fluoresces weakly in the presence of water or single strands of DNA. The green dye detects double-stranded DNA generated during PCR. It is also used to detect DNA in gel electrophoresis. Single-Drop Fluorimeter The Single-Drop Fluorimeter is a device used to measure fluorescence. The base of the fluorimeter houses a slide holder specialized to hold a rectangular glass slide that has a hydrophobic coating. This coating allows water to collect in a droplet on the surface that will be used to measure the fluorimeter's light source, a blue LED light. The base also allows a camera to sit in front of the slide. Finally, a light box is placed over the base that encloses the fluorimeter and prevents light from outside sources from entering, making the droplet and LED more visible for the camera to capture. How the Fluorescence Technique Works Fluorescence is a process in which the emission of light from a sample droplet is measured in order to collect data. For this experiment specifically, a blue LED is passed through a droplet containing a sample of DNA and SYBR Green dye which will fluoresce when interacted together. This LED light excites the molecules within the droplet causing the droplet to emit light in a more focused wavelength which can be measured visibly when captured in a picture using a camera. On a more theoretical level, the light given off by the droplet is measured by the equation of fluorescence, which states that the product of the intensity of the LED, the absorption coefficient and molar concentration of SYBR Green dye, and a value of quantum efficiency, equals the fluorescent intensity. This fluorescent intensity is then related to the concentration of DNA in the droplet when removing the background fluorescence of non-reacting molecules (Week10 Lab Handout-BME 100). Procedure Smart Phone Camera Settings Type of Smartphone: iPhone 5s Flash: off ISO setting: n/a White Balance: n/a Exposure: n/a Saturation: n/a Contrast: n/a Calibration Distance between the smart phone cradle and drop = 5 cm Solutions Used for Calibration Calf Thymus DNA (μg/mL) SYBR Green Dye Volume (μL) 2X DNA Solution Volume (μL) Final DNA Concentration (ng/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 blank Placing Samples onto the Fluorimeter Step One: Insert a glass slide into the fluorimeter with the "smooth side" facing down. Step Two: Extract X ml of SYBR green fluid and place it on the sample slide between the First and Second row on the middle dots. Step Three: Extract and place X ml of the sample concentration that is to be tested on the same fluid that was used int he previous step. Step Four: After each photo has been taken, remove the sample fluid and dispose of it as instructed. Step Five: Move the slide into position in such that the blue light is directed onto the middle of the next 2 dots that have not been used. Step Six: Repeat steps 1-5 until all samples are tested. Remember to use a new slide when each dot has been used on the slide. Data Analysis Representative Images of Samples Positive Signal Image J Values for All Samples Calf Thymus DNA Concentration (FINAL), μg/mL ' AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDEN - BACKGROUND 2.5 image 1 57175 91.476 5017524 101447 4916077 2.5 image 2 56606 89.232 5051060 158329 4892731 2.5 image 3 57980 94.259 5465111 93588 5371523 1 image 1 54792 92.851 5087471 107397 4980074 1 image 2 58002 77.445 4491953 90963 4400990 1 image 3 53835 74.786 4026096 92974 3933122 0.5 image 1 51093 77.155 3942062 82723 3859339 0.5 image 2 49822 83.888 4179470 75890 4103580 0.5 image 3 52412 86.055 4510312 90407 4419905 0.25 image 1 49036 85.196 4177657 80663 4096994 0.25 image 2 54926 88.903 4883089 95912 4787177 0.25 image 3 49940 78.76 3933264 93143 3840121 0.125 image 1 46861 54.974 2576142 88321 2487821 0.125 image 2 51066 53.409 3238055 89218 3148837 0.125 image 3 56664 87.337 4948882 100275 4848607 0 image 1 47032 46.163 2171138 76774 2094364 0 image 2 51824 49.35 2557534 77391 2480143 0 image 3 46976 57.165 2685374 178179 2507195 Fitting a Straight Line Disclaimer: It is also worth noting that the RAWINTDEN values are much lower than the theoretical values. In addition, there is less than optimal change of the RAWINTDEN values between the concentrations of the DNA. Even though the difference is minute, it is still measurable, and these data points can still be used for a calibration as long they are consistent with the values in the next experiment using the actual PCR DNA.