BME103 s2013:T900 Group4 L2

BME 103 Spring 2013	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Kinjal Ahir : Procedure Name: Tuan Phan : Background Information Name: student Name: student Name: student LAB 2 WRITE-UP Background Information SYBR Green Dye [A short summary describing SYBR green dye] SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light (λmax = 497 nm) and emits green light (λmax = 520 nm). The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower performance. SYBR green can also stain RNA with a lower performance than DNA. SYBR green finds usage in several areas of biochemistry and molecular biology. It is used as a dye for the quantification of double stranded DNA in some methods of real time PCR. It is also used to visualize DNA in gel electrophoresis. Higher concentrations of SYBR green can be used to stain agarose gels in order to visualize the DNA present. In addition to labeling pure nucleic acids SYBR green can also be used for labeling of DNA within cells for flow cytometry and fluorescence microscopy. In these cases RNase treatment may be required to reduce background from RNA in the cells. http://en.wikipedia.org/wiki/SYBR_Green_I Single-Drop Fluorimeter [A description of the single-drop fluorimeter device. Add a PHOTO for bonus points] Fluorimeter is a device that is used to measure fluorescence. How the Fluorescence Technique Works [In your own words, a summary of the information from page 9 of the worksheet] Procedure Smart Phone Camera Settings [Iphone 4] Flash: Off ISO setting:NA White Balance: NA Exposure: NA Saturation: NA Contrast: NA Calibration Photo of Fluorimeter Put your phone on the cradle at a right angle Then fix the height of the fluorimeter so that the camera can take picture of the drop on sideways Distance between the smart phone cradle and drop = Solutions Used for Calibration [See worksheet page 5] Calf Thymus DNA solution concentration (microg/ML) Volume of the 2x DNA solution (uL) Volume of the SYBR GREEN Dye Solution (uL) Final DNA concentration PicoGreen Assay (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, set your phone Step two,Pick up DAN, gloves, lab coat, glass slide, buffer tubes, sync green I tube, and DNA standard Step three, Slide the glass slide into fluorimeter Step four, Drop 80 micro liters of sample of the solution where the light is flashing Step five, Look to see if the drops forms a ball and doesn't spread around Step six, Place the box around it and take a picture Data Analysis Representative Images of Samples [Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA] [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 [See worksheet page 5] Trial DNA Concentration (μg/mL) Area Mean Pixel Value RAWINTDEN (drop) RAWINTDEN (background) 1 0 10,906 21.672 236,351 70,338 1 0.25 10,044 35.971 361,290 42,572 1 0.5 10,562 84.274 890,103 52,974 1 0.5 10,452 50.546 528,303 50,012 1 1 10,350 115.226 1,192,594 39,995 1 2 10,914 193.145 2,107,989 46,923 1 5 10,282 142.563 1,465,554 55,342 2 0 10,808 50.412 544,856 71,106 2 0.25 10,510 51.038 536,412 39,795 2 0.5 10,688 78.799 842,203 57,075 2 1 10,432 90.965 948,944 52,390 2 2 10,103 152.075 1,536,481 61,000 2 5 10,440 115.674 1,207,635 51,873 3 0 10,131 37.264 377,520 66,289 3 0.25 10,448 61.987 647,645 48,887 3 0.5 10,762 64.475 693,875 40,978 3 1 10,465 74.835 783,144 47,549 3 2 10,188 155.63 1,585,554 43,186 3 5 10,672 72.312 771,711 47,142 [Add more rows as needed] Fitting a Straight Line [Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 8]