BME100 f2013:W900 Group10 L5: Difference between revisions
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'''SYBR Green Dye'''<br> | '''SYBR Green Dye'''<br> | ||
SBYR green dye is a cyanine dye used as a nucleic acid stain. | 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'''<br> | '''Single-Drop Fluorimeter'''<br> | ||
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. | |||
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'''How the Fluorescence Technique Works'''<br> | '''How the Fluorescence Technique Works'''<br> | ||
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. | 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 data 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. | |||
Revision as of 16:32, 9 November 2013
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
LAB 5 WRITE-UPBackground InformationSYBR 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.
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.
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 data 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.
ProcedureSmart Phone Camera Settings
[Instructions: If you used an additional phone, describe the other type of smart phone you used and how you adjusted the camera settings, if applicable. If not, delete this part]
[Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.]
[Instructions: See worksheet page 6.]
Data AnalysisRepresentative 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.]
[Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]
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