BME100 f2013:W900 Group10 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 Name: Joslin Jose Name: Barrett Anderies Name: student Name: student Name: student Name: student LAB 5 WRITE-UP Background Information SYBR Green Dye SBYR green dye is a cyanine dye used as a nucleic acid stain. When bound to DNA it absorbs blue light and emits green light. The stain binds to double stranded DNA at very high levels and binds to single stranded DNA at much lower levels. This allows us to measure the amount of double stranded DNA while getting minimal noise on our signal from the presence of single stranded DNA. It can also stain RNA at lower levels, but this is not important for our experiment (in which we know our sample does not contain RNA). It is also the most sensitive stain available for detecting double stranded DNA during PCR. Single-Drop Fluorimeter This device is used to excite the stain molecules in order to generate the signal (green light) for us to capture. The single-drop fluorimeter is designed to hold a single drop of the sample and pass the wavelength of light required to excite the stain molecules through the sample. This light is high energy (short wavelength -- blue/ultraviolet) light. Pervious: This device is used to measure parameter of fluorescence such as intensity and wavelength and the display of emission spectrum after excitation by a certain spectrum of light. It detects and measures ultraviolet radiation by determining the amount of fluorescence that it produces from a phosphor fluorometric. [Instructions: A description of the single-drop fluorimeter device. Add a PHOTO for bonus points] How the Fluorescence Technique Works A droplet is placed on the hydrophobic slide which allows the droplet to hold its spherical shape. The sample droplet is then exposed to an ultraviolet light light beam to excite the stain molecules which proceed to emit green light (our signal). We capture this signal (green light) with our smartphone camera. In theory, the amount of signal captured by our camera should be proportional to the concentration of DNA in the sample (the pictures must be filtered so that only the amount of green light captured is taken into consideration). Therefore, once we have calibrated our camera with known concentrations of DNA we should be able to compare signal strengths (green light emittance) from unknown DNA concentration samples with our calibration data to accurately estimate the DNA concentration in that sample. Previous: A droplet is placed on the hydrophobic slide which allows the droplet to hold its spherical shape. Then the slides are exposed to two light beams from either sides which decreases the surrounding noise. The upper beam passes through a filter that passes through a sample. The lower beam is passed through an attenuator and tries to match the fluorescent power given off from the sample. Both light is detected by separate transducers and turned into an electrical signal. [Instructions: In your own words] Procedure Smart Phone Camera Settings [Instructions: The type of smart phone you used and how you adjusted the camera settings, if applicable (see worksheet page 4).] Type of Smartphone: Samsung Galaxy SIII Flash: OFF ISO setting: 800 White Balance: Auto Exposure: +2 (maximum) Saturation: Auto (no manual options) Contrast: Auto (no manual options) Calibration [Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.] The camera was placed upright in the supplied stand. The camera lens was positioned 6cm from the droplet in order to get the closest view possible while still being within the focus range of the camera. The height of the fluorimeter was adjusted so that the lens of the camera was at the same height as the droplet to ensure a full side view. Distance between the smart phone cradle and drop = 6cm [Instructions: See worksheet page 6.] Solutions Used for Calibration [Instructions: See worksheet page 6.] [Add more rows as needed]Placing Samples onto the Fluorimeter [Instructions: Step one, in your OWN words] [Instructions: Step two, in your own words] [Instructions: Step three, in your own words] [Instructions: Step etc., in your own words] 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.] TABLE GOES HERE Fitting a Straight Line [Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]