BME100 s2014:W Group14 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: Jose Duran Name: Shawn Garcia Name: Christian Keefer Name: Karthik Puncha Name: Austin Tielke LAB 5 WRITE-UP Background Information SYBR Green Dye [Instructions: A short summary describing SYBR green dye] Single-Drop Fluorimeter [Instructions: A description of the single-drop fluorimeter device. Add a PHOTO for bonus points] How the Fluorescence Technique Works [Instructions: In your own words] Procedure Smart Phone Camera Settings Type of Smartphone: Apple iPhone 5S Flash: Off ISO setting: Auto White Balance: Auto Exposure: High Saturation: High Contrast: Low Calibration The camera was set up by placing the iPhone 5S in the holding compartment of the lab equipment. It was measured to be 6 centimeters away from the origin of the light emission. The settings (stated above) were adjusted; the height of the fluorimeter was adjusted in order to achieve a perfect side-angle perspective of the "beach ball" shaped liquid drop. Distance between the smart phone cradle and drop = 8 centimeters Solutions Used for Calibration Initial Concentration of 2X Calf Thymus DNA solution (μg/mL) Volume of the 2X DNA solution (μL) Volume of the SYBR GREEN I Final DNA concentration in SYBR Green I solution (μg/mL) 5 80 80 2.5 2 80 80 1.0 1 80 80 0.5 0.5 80 80 0.25 0.25 80 80 0.125 0 80 80 0 Placing Samples onto the Fluorimeter Before placing samples onto the fluorimeter, turn on the Blue LED light & have the camera on the smartphone ready. Insert the glass sampling surface, with the smooth side facing down. Position the glass sampling surface so that the light emits between the first two rows of the glass. Place an 80 μL drop of SYBR Green I in the middle of the first two rows. Swap out the pipette tip with a new tip. Place an 80 μL drop of a specifically concentrated Calf Thymus DNA solution onto the top of the previously placed SYBR Green I drop. Every three 160 μL sample drops will vary in concentrated Calf Thymus DNA solution. In order to place a new sample drop, remove the 160 μL solution, move the glass sampling surface to the next two available rows, and repeat the process of placing two new 80 μL drops onto each other (as stated before). Data Analysis Representative Images of Samples Sample w/o DNA Sample w/ DNA Image J Values for All Samples Final DNA Concentration in SYBR Green I Solution (μg/mL) Area (pixels) Mean Pixel Value RAWINTDEN of the Drop RAWINTDEN of the Background RAWINTDEN Drop - RAWINTDEN Background 0.5 60452 85.151 4421613 187151 4234462 0.5 59871 84.124 4521561 175412 4346149 0.5 60241 82.153 4159878 179854 3980024 1.0 63157 120.451 5904302 195454 5708848 1.0 64120 124.021 6048248 187945 5860303 1.0 63785 117.879 6248451 198454 6049997 2.5 73482 164.546 8558810 212416 8346394 2.5 74125 170.215 8821541 205451 8616090 2.5 73956 177.154 8258495 215411 8043084 0.0 56235 7.546 3201562 167842 3033720 0.0 56345 5.241 3158472 154952 3003520 0.0 57120 6.542 3584956 151231 3433725 0.125 57325 22.145 1124652 165451 959201 0.125 57694 24.564 1413586 167851 1245735 0.125 57120 27.156 1248412 170245 1078167 0.25 58503 78.151 3952152 168672 3783480 0.25 59871 84.4561 3754546 171541 3583005 0.25 59212 80.541 4132142 172164 3959978 Fitting a Straight Line PCR Results Summary TUBE LABEL Volume of the DILUTED PCR Product solution (µL) Volume of the SYBR GREEN I INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS " " AVERAGE INTDENS VALUE Dye solution (µL) A00 80 80 17514607.24 16358382.85 13105657.98 15659549.36 A01 80 80 7527051.87 5439907.836 5817272.88 6261410.862 A02 80 80 5780187 5264154.775 7535894.664 6193412.146 A03 80 80 7493409.77 6729272.54 6821746.185 7014809.498 B00 80 80 1885223.456 1780211.712 1991211.874 1885211.816 B01 80 80 6901246.104 8036257.608 9159860.332 8032454.681 B02 80 80 8841431.624 10061646.59 9186306.417 9363128.21 B03 80 80 10232940.21 11665080.57 10564810.97 10820943.92 PCR Product Tuble Label Average INTDENS Value PCR Product Concentration (μg/mL) Corrected PCR Product Concentration (μg/mL) A00 15659549.36 10.53189465 0.877657887 A01 6261410.862 2.952750695 0.246062558 A02 6193412.146 2.897913021 0.241492752 A03 7014809.498 3.56033024 0.296694187 B00 1885211.816 -0.576442084 -0.04803684 B01 8032454.681 4.38101184 0.36508432 B02 9363128.21 5.454135653 0.454511304 B03 10820943.92 6.629793484 0.55248279 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 0.877657887 μg/mL Your negative control PCR result was -0.04803684 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 26734: Patient 22707: 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 26734: Patient 22070: