BME100 f2013:W1200 Group6 L5

BME 100 Fall 2013	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 Jenny Chen Tracy Lopez Nayobe Bivins Alex Bugarin Nicholas Kilpatrick LAB 5 WRITE-UP Background Information SYBR Green Dye The SYBR green dye is an important component of the fluorescence technique for detecting the amplified target DNA during a PCR (polymerase chain reaction) experiment. It is a dye that detects the presence of dsDNA (double-stranded DNA) and only binds to its structure; after being bound to dsDNA (double-stranded DNA) it later emits a bright green color. In contrast, the SYBR green dye produces a weak fluorescence whenever it binds to other molecules such as water or single-stranded DNA. Single-Drop Fluorimeter [Instructions: A description of the single-drop fluorimeter device. Add a PHOTO for bonus points] The fluorimeter is a laboratory instrument that serves as a DNA or protein quantification device because it accurately measures the amount of fluorescence present in a sample in widely used applications such as PCR. The Fluorimeter that was used in this lab was a black box that has a space in the middle. The space in the middle was used to easily slide in the hydrophobic glass slides. The Hydrophobic slides were used so that the substance could stay as a bubble due to the hydrophobic properties. There is a switch on the side of the box and when it is flipped on the fluorimeter transmits a blue LED light. That blue light goes through the substance that is on the glass slide.*Need to add more sentences* How the Fluorescence Technique Works The Fluorescence technique [Instructions: In your own words] Procedure Smart Phone Camera Settings Type of Smartphone: Samsung Galaxy S4 Flash: None ISO setting: 800 ISO White Balance: Auto Exposure: 2.0 Saturation: N/A Contrast: N/A No additional Smartphone was used Calibration The camera was placed in the cradle in front of the fluorimeter and the camera application selected. There was a box covering the whole set to not have any outside source of light. Based on the view of the blue light, the camera position was adjusted until the droplet would be large and centered on the screen. The distance between the drop and cradle was then measured. With a timer enabled on the camera we were able to close the lid on the box to create a closed environment. [Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.] Distance between the smart phone cradle and drop = 9.7 cm [Instructions: See worksheet page 6.] Solutions Used for Calibration Calf Thymus DNA solution concentration (µg/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN I Dye solution (µL) Final DNA concentration in SYBR Green I Assay (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 blank Placing Samples onto the Fluorimeter Set the micropipet to 80 microliters and put on a new tip. Put 80 microliters of SYBR GREEN I solution in between the first two rows of the slide, with the blue light passing through the droplet. Using a new tip, put 80 microliters of one of the solutions into the same droplet. Repeat this process with all the designated solutions, using the micropipet set at 160 microliters to remove each sample in between and moving the slide forward so the blue light passes through the droplet each time. Data Analysis Representative Images of Samples [Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA] [Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with DNA (positive signal)] Image J Values for All Samples [Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.] Calf Thymus DNA Concentration (FINAL), ìg/mL ' AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDEN - BACKGROUND 2.5 image 1 155640 156.535 24363157 129814 24233343 2.5 image 2 146804 144.342 21189975 52893 21137082 2.5 image 3 139396 143.53 20007563 98923 19908640 1 image 1 168304 105.241 17712539 154616 17557923 1 image 2 160336 108.046 17323630 13078 17310552 1 image 3 166560 108.104 18005867 51001 17954866 0.5 image 1 154902 63.077 9770677 53939 9716738 0.5 image 2 144268 65.413 9437067 86282 9350785 0.5 image 3 157834 64.486 10178120 54290 10123830 0.25 image 1 157236 46.207 7265369 58401 7206968 0.25 image 2 174990 46.154 8076559 85170 7991389 0.25 image 3 153700 46.804 7193718 54061 7139657 0.125 image 1 176180 21.629 3810623 87020 3723603 0.125 image 2 171193 19.744 3379991 34984 3345007 0.125 image 3 175499 17.743 3113945 128292 2985653 0 image 1 157512 45.899 7229573 61120 7168453 0 image 2 140430 52.219 7333154 45801 7287353 0 image 3 150408 45.277 6809958 41408 6768550 Fitting a Straight Line [Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]