BME100 f2013:W900 Group10 L5

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Procedure)
(Procedure)
Line 68: Line 68:
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.
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
+
* Distance between the smart phone camera lens and drop = 6cm
'''Solutions Used for Calibration'''  
'''Solutions Used for Calibration'''  
Line 89: Line 89:
'''Placing Samples onto the Fluorimeter'''
'''Placing Samples onto the Fluorimeter'''
-
# ''[Instructions: Step one, in your OWN words]''
+
# Place a clean slide with the hydrophobic side up into the slot in the Fluorimeter.
-
# ''[Instructions: Step two, in your own words]''
+
# Turn on the Fluorimeter.
-
# ''[Instructions: Step three, in your own words]''
+
# Use a micro-pipette to transfer 80 µL of SYBR Green I Dye and 80 µL of sample (either water or DNA solution) onto the slide to form a single droplet.
-
# ''[Instructions: Step etc., in your own words]''
+
# Move the slide until the droplet is directly in line with the blue light beam (if not so already).
 +
# Position camera at the predetermined position (see "Calibration") and set a countdown timer on the camera.
 +
# Focus the camera on the droplet and start the countdown timer.
 +
# Cover the entire apparatus with the supplied box to minimize external light and wait for the camera to capture the picture.
 +
# Remove the box, open the recently captured picture and rename it to something that represents the sample DNA concentration and picture number.
 +
# Remove the 160 µL droplet from the Fluorimeter slide with a micro-pipette and remove any remaining liquid with a paper towel.
 +
# Repeat the above steps two more times for each sample to get three pictures of three different droplets of the same combination of SYBR Green I Dye and sample.
<br>
<br>

Revision as of 16:25, 10 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
Image:BME494_Asu_logo.png

Contents

OUR TEAM

Name: Joslin Jose
Name: Joslin Jose
Name: Barrett Anderies
Name: Barrett Anderies
Name: student
Name: student
Name: student
Name: student
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 (~497nm) of light required to excite the stain molecules through the sample.

[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.

[Instructions: In your own words]



Procedure

Smart Phone Camera Settings

  • 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 camera lens and drop = 6cm

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 (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

  1. Place a clean slide with the hydrophobic side up into the slot in the Fluorimeter.
  2. Turn on the Fluorimeter.
  3. Use a micro-pipette to transfer 80 µL of SYBR Green I Dye and 80 µL of sample (either water or DNA solution) onto the slide to form a single droplet.
  4. Move the slide until the droplet is directly in line with the blue light beam (if not so already).
  5. Position camera at the predetermined position (see "Calibration") and set a countdown timer on the camera.
  6. Focus the camera on the droplet and start the countdown timer.
  7. Cover the entire apparatus with the supplied box to minimize external light and wait for the camera to capture the picture.
  8. Remove the box, open the recently captured picture and rename it to something that represents the sample DNA concentration and picture number.
  9. Remove the 160 µL droplet from the Fluorimeter slide with a micro-pipette and remove any remaining liquid with a paper towel.
  10. Repeat the above steps two more times for each sample to get three pictures of three different droplets of the same combination of SYBR Green I Dye and sample.


Data Analysis

Representative Images of Samples

The picture below shows the control case where no DNA is present in the sample

Image: BME100_Group_10_Drop_Without_DNA.png

The picture below shows a test case where DNA is present in the sample

Image: BME100_Group_10_Drop_With_DNA.png


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]



Personal tools