BME100 s2014:W Group8 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: Gabriela Coote Name: Brianna Snyder Name: Ethan Wheeler Name: Chloe Houlihan Name: Yesenia Meadows LAB 5 WRITE-UP Background Information SYBR Green Dye The SYBR Green Dye is a small molecular dye that, when in the presence of dsDNA, will become very fluorescent but when in the presence of water or ssDNA, will not become very fluorescent. Single-Drop Fluorimeter The single-drop fluorimeter is a black box with a cradle on top for the glass slide that will have a drop on it. Towards the front of the cradle there is a blue LED light that can be toggled on or off with a switch. Image 1 shows the top view of the fluorimeter. How the Fluorescence Technique Works Fluorescence microscopy has been used in order to highlight parts of the lab experiment that is being recorded. The method in this lab has been used in order to test the concentration of DNA within a sample. We used SYBR green for the experiment. The SYBR green in the sample binds to the sample DNA and, once bound, fluoresces giving off green light when illuminated by blue light. The green light that is emmited from the drop of DNA/SYBR green is indicative of the concentration of DNA in the sample. The reason why we use a blue LED is because it intensified the florescence event. A lab should use SYBR green to dye the DNA because it is environmentally friendly and also other dyes (that are soluble in water) would not become florescent when the LED light hits the sample. Procedure Smart Phone Camera Settings Type of Smartphone: iPhone 4 Flash: No flash ISO setting: 800 White Balance: Auto Exposure: Highest Saturation: Highest Contrast: Lowest Calibration To set up the system, place the smartphone on the cradle at a right angle. Adjust the fluorimeter height using plastic trays so that the camera takes a picture of the drop sideways. Change the distance of the smart phone from the fluorimeter so that the phone can take a clear image. Image 2 shows the basic set up of the system. * Distance between the smart phone cradle and drop = 11.52 cm Solutions Used for Calibration [Instructions: See worksheet page 6.] Placing Samples onto the Fluorimeter Place a blank side into the Fluorimeter Turn on the blue led "excitation light" Place 80 microliters of SYBR Green dye in the middle of the first two rows of the slide Place 80 microliters of the calf thymus on top of the SYBR Green dye Place the camera in the cradle so it is able to take a picture of the side of the drop with the timer and flash on Record the distance between the phone and the drop Take picture Data Analysis Representative Images of Samples Image 3 shows the drop without DNA. Image 4 shows the drop with DNA. Image J Values for All Samples Calibration Final DNA concentration in SYBR Green I solution (µg/mL) AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND 1. 2.5 160 150.369 24059 1194 2. 2.5 826 230.78 190624 10613 3. 2.5 860 235.206 202277 30313 4. 1.0 471 171.968 80997 10094 5. 1.0 502 165.169 82915 14455 6. 1.0 474 176.766 83787 19964 7. 0.5 615 119.208 73313 20552 8. 0.5 816 116.776 95289 9187 9. 0.5 651 123.55 80431 5511 10. 0.25 428 74.701 31972 12575 11. 0.25 521 76.896 40063 17322 12. 0.25 540 82.072 44319 8081 13. 0.125 384 51.302 19700 6656 14. 0.125 406 48.613 19737 9009 15. 0.125 412 53.32 21968 5425 16. 0.0 512 22.373 11455 4725 17. 0.0 474 22.532 10680 9211 18. 0.0 471 22.541 10617 6465 PCR Products PCR Product TUBE LABEL Volume of the DILUTED PCR Product solution (µL) Volume of the SYBR GREEN I Dye solution (µL) ' INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS' ' AVERAGE INTDENS VALUE 1. 8PC 80 80 182084 152527 153601 162737 2. 8λ1 80 80 59485 40356 39911 46584 3. 8λ2 80 80 48082 56735 64465 56427 4. 8λ3 80 80 50058 49921 50493 50157 5. 8NC 80 80 56801 68031 60370 61734 6. 8θ1 80 80 238412 217854 188389 214885 7. 8θ2 80 80 231847 235892 212734 226824 8. 8θ3 80 80 220105 153560 158086 177250 Concentration of PCR Product (DNA) PCR Product TUBE LABEL Volume of the DILUTED PCR Product solution (µL) Volume of the SYBR GREEN I Dye solution (µL) INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS ' ' AVERAGE INTDENS VALUE 1. 8PC 80 80 182084 152527 153601 162737 2. 8λ1 80 80 59485 40356 39911 46584 3. 8λ2 80 80 48082 56735 64465 56427 4. 8λ3 80 80 50058 49921 50493 50157 5. 8NC 80 80 56801 68031 60370 61734 6. 8θ1 80 80 238412 217854 188389 214885 7. 8θ2 80 80 231847 235892 212734 226824 8. 8θ3 80 80 220105 153560 158086 177250 Fitting a Straight Line Image 5 shows the best fit line of the data from the calibration table.