BME100 f2013:W1200 Group14 L5

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png 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
BME494 Asu logo.png

OUR TEAM

Name: Vaasavi Sundar
Name: Hannah Brutsche
Name: Minh Pham
Name: Brianna Schilling
Name: Ashley Powell


LAB 5 WRITE-UP

Background Information

SYBR Green Dye

The SYBR green dye I is used as a stain for nucleic acid testing. SYBR green dye I binds to double stranded DNA and is very fluorescent when bound to double stranded DNA. SYBR Green I absorbs blue light, and emits green light. That is why the blue LED light is used to visualize the fluorescence. Furthermore, SYBR Green Dye I also emits fluorescence when binding to PCR products that contain double-stranded DNA, or dsDNA. However, the SYBR green dye I does not illuminate as well with water or single stranded DNA, which makes it a highly useful product when measuring the amount of PCR product that was created.

Single-Drop Fluorimeter

CAM00424.jpg
This instrument detects fluorescence, which is proportional to the amount of fluorescent material, and the amount of the molecule that is detected. The single drop fluorimeter works by measuring the intensity of the excitation of the light, and the wavelength distribution of this light throughout the drop. Optical caustics is a key idea that is applicable in the use of the single drop fluorimeter.

How the Fluorescence Technique Works
The surface of the slide is covered with extremely water-resistant Teflon-- however, the slide also has separate sections which are simply glass. This allows for the blue LED light pay special focus to the droplet, which should increase the intensity and the degree to which fluorescence can be detected. The difference in the surface of the slide allows for the SYBR green dye to bind with DNA and form a substance that ends up going closer to the surface. The use of the lightbox makes sure that it is easier to detect fluorescence within the drop. This is repeated for each calibration solution, which ranges in the concentration of DNA from 0 mg/mL to 2mg/mL. The images from each of these solutions is analyzed by ImageJ, to find the RAW INTDEN of both the droplet and the background.

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone4s
    • Flash:none.
    • ISO setting: 800
    • White Balance: Auto
    • Exposure:N/A
    • Saturation: N/A
    • Contrast: N/A


Calibration


CAM00422.jpg

  • Distance between the smart phone cradle and drop = 6cm


Solutions Used for Calibration

Calf Thymus DNA solution concentration (microg/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. Put a glass slide into the fluorimeter, making sure that the light is in-between two rows.
  2. Place a 80 microliter drop of SYBER Green in the middle of the first two rows using the pipette. (Make sure it looks like a ball)
  3. Next add 80 microliters of the calibration solutions listed.
  4. Align a camera at the edge of the slide to take an image of the drop.
  5. Place a timer on the camera and take 3 images, immediately close the lid to ensure that little to no light enters the box.
  6. Use the micropipette to remove the drop and dispose of it and the tip.



Data Analysis

Representative Images of Samples

This is the image of the droplet without DNA in it. BME100samplewithoutDNA.jpg

This is the image of the droplet with DNA in it. BME100samplewithDNA.jpg


Image J Values for All Samples

Calf Thymus DNA Concentration (FINAL), μg/mL ' AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND
2.5 image 1 116820 115.611 13505698 585970
2.5 image 2 117868 113.553 13384211 456043
2.5 image 3 1084 114.81 124454 6409
1 image 1 143848 101.92 14661050 376378
1 image 2 129212 106.759 13794585 1563279
1 image 3 1060 116.125 123093 9701
0.5 image 1 1276 73.624 93944 4321
0.5 image 2 1292 76.67 99058 4763
0.5 image 3 1328 72.082 95725 5579
0.25 image 1 1857 26.037 48351 1496
0.25 image 2 1866 28.88 53891 1788
0.25 image 3 2058 24.375 50163 2002
0.125 image 1 163132 12.138 19800788 10506
0.125 image 2 156588 8.62 1349719 68339
0.125 image 3 146880 6.912 11888 1809
0 image 1 165796 16.31 2704074 7666
0 image 2 173668 7.418 1288196 15942
0 image 3 175244 7.8 1366883 6792


Fitting a Straight Line

BME100scatterplot.jpg