BME100 s2014:T Group9 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: Cassaundra Kinnear Name: Katherine Underwood Name: Kyle Queen Name: Mohammed Binsheliail LAB 5 WRITE-UP Background Information SYBR Green Dye SYBR green dye is used as a nucleic acid stain and binds to DNA. The result is a DNA-dye-complex that absorbs blue light and emits green light. This dye can also bind to single-stranded DNA and RNA with a lower performance. Single-Drop Fluorimeter A single-drop fluorimeter is made up of a sturdy black cover used to block out the light. There is a phone cradle used o hold the camera while taking pictures. Inside the black cover there is a platform to hold the slides. There is also an LED light placed around the slides. This light emits a blue light used to see the SYBR green dye. How the Fluorescence Technique Works The blue LED light shines on the DNA-dye samples. The amount of DNA in each sample is measured based on the concentration of the green light being emitted from the sample. Procedure Smart Phone Camera Settings Type of Smartphone: iPhone 4s Flash: off ISO setting: N/A White Balance: N/A Exposure: N/A Saturation: N/A Contrast: N/A Calibration The camera was set on a cradle eye level with the sample. We set the camera with a timer so there would be no other light than the LED light the green glow coming from the sample. We clicked the button and closed the flap of the outer black case, then the camera took the picture of the sample. Distance between the smart phone cradle and drop = 8 cm Solutions Used for Calibration Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN I Dye solution (µL) Final DNA concentration in SYBR Green I solution (µg/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 0 Placing Samples onto the Fluorimeter Place the fluorimeter into the box Place the camera in the cradle Adjust the height of the fluorimeter with the trays so the camera will see the drop at eye level Insert the slide into the fluorimeter Adjust the distance between the camera and the slide to be as close as possible without being blury Place an 80 microliters drop of SYBR Green in between the first two rows of the slide Add 80 microliters of the indicated DNA solution to the drop of SYBR Green Align the sample and the blue LED light Click the timer on the camera and close the lid for the picture Take one more picture, for a total of two pictures, without moving the camera Data Analysis Representative Images of Samples Image J Values for All Samples Concentration Area Mean IntDen RawIntDen RawIntDen Back 0 89824 22.134 1988142 1988142 4075684 106024 25.339 2686504 2686504 3520178 244420 39.829 9735070 9735070 4252614 0.25 232620 54.65 12712759 12712759 1470699 213096 60.944 12986996 12986996 1545487 230408 43.885 10111560 10111560 1460134 0.5 316088 86.678 27397838 27397838 5561052 263648 111.592 29421095 29421095 5410757 228141 134.47 30678127 30678127 1187005 1 275068 121.335 33375368 33375368 1276411 285964 116.944 33441633 33441633 1345189 99532 113.984 11345102 11345102 1352381 2 101468 114.739 11642363 11642363 921896 101884 125.741 12810987 12810987 957122 109756 115.55 12682322 12682322 1114350 5 108804 173.976 18929237 18929237 654847 109360 120.332 13159480 13159480 796070 106772 111.949 11952999 11952999 918322 Excel-to-Wiki Converter. Tube A Tube B Tube C Tube D Tube E Tube F Tube G Tube H 2308221 4130155 5069909 5325157 11018266 14630361 17579974 21359877 4078569 4773563 5282577 5753055 11153155 16780232 18268541 21671092 4107249 5055747 5291947 7644472 13454922 17148850 20260575 22388687 Fitting a Straight Line PCR Results Summary 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 ____ μg/mL Your negative control PCR result was ____ 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 _____ : Patient _____ : 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 _____ : Patient _____ :