BME100 f2013:W900 Group14 L5

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OUR TEAM

Name: Rachael Brard
Name: Austin Davis
Name: Andrew Cable
Name: Omar Eltohamy


LAB 5 WRITE-UP

Background Information

SYBR Green Dye
SYBR green dye is a nucleic acid stain that turns fluorescent in the presence of double stranded DNA. It is often used to bind with double-stranded DNA molecules. It can also bind with one-stranded DNA molecules as well as RNA molecules, but with less performance.

Single-Drop Fluorimeter
The device has a lowered area where the slide goes, and a blue beam of light is projected across this area. The toggle on the side of the Fluorimeter controls the light. A drop of DNA and SYBR dye is combined and placed upon the slide and the slide is adjusted so that the drop is in the middle of the first two clear dots and the light squarely hits the drop.

Group 14 Fluorimeter.JPG


How the Fluorescence Technique Works
The DNA and SYBR dye drop is repelled by the superhydrophobic Teflon slide so that it sits on the surface. The beam of blue LED light hits the drop and a green fluorescence is produced due to the SYBR green dye. This fluorescence makes the solution much easier to observe and collect data from. The fluorescence also helps determine the double-stranded DNA molecules that are being bonded as well.

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy S III
    • Flash: Off
    • ISO setting: N/A
    • White Balance: N/A
    • Exposure: N/A
    • Saturation: N/A
    • Contrast: N/A


Calibration

Place smartphone in cradle so that the lens is set up straight in front of the drop on the slide. Measure the distance between the phone and the drop with a ruler and place the dark box over the system. Phone was placed in level with the height of the drop, in order to get a more clearer picture of the drop and the fluorescence. If needed, place a pencil or small object on the stand without interfering with the phone in order to keep the stand steady and immovable. Set the camera on the phone, and place a timer on the camera for the best results, as this helps eliminate any light that may enter in the box. Close the lid and block out as much light as possible for the best picture. If needed, take multiple pictures until the best one is taken. Record the pictures with proper amount of concentration of SYBR Green Dye.

Group 14 Calibration.jpg


  • Distance between the smart phone cradle and drop =

5 cm


Solutions Used for Calibration '

' Placing Samples onto the Fluorimeter
  1. Pumping the 80 microliters of DNA onto the fluorimeter
  2. Add the SYBR Green to the solution of DNA on the fluorimeter
  3. Place the slide with the SYBR Green and DNA solution onto the fluorimeter
  4. Adjust phone with camera on the cradle for optimum photo
  5. Put the shadow box over the fluorimeter and solution then take pictures
  6. Ensure lid was closed and all light possible was blocked out before taking picture
  7. Record picture with name of contraction of SYBR Green Dye (ex: Calibration Drop .5)
  8. Remove the stand, phone, as well as the shadow box from above the flourimeter
  9. Pull out flourimeter and wipe it clean with paper towel so no drop exists
  10. Repeat for all concentrations of SYBR Green Dye until all concentration are complete

Data Analysis

Representative Images of Samples

Group 14 NO DNA.png
This is our sample shown on Image J with zero DNA added.

Group 14 SOME DNA.png
This is our sample shown on Image J with 2.5 micrograms/mL of DNA added.


Image J Values for All Samples


Concentration of DNA Volume of DNA Solution Volume of SYBR Green Final Concentration of DNA
0 ug/uL 80 uL 80 uL 0 ug/uL
.25 ug/uL 80 uL 80 uL .125 ug/uL
.5 ug/uL 80 uL 80 uL .25 ug/uL
1 ug/uL 80 uL 80 uL .5 ug/uL
2 ug/uL 80 uL 80 uL 1 ug/uL
5 ug/uL 80 uL 80 uL 2.5 ug/uL
DNA concentration Area (in pixels) Mean Pixel Value RAWINTDEN
0 26077 40.368 1052680
0 (noise) 26077 0.017 446
0.25 29376 32.13 943842
0.25 (noise) 29376 0.014 407
0.5 23256 65.424 1521500
0.5 (noise) 23256 0.123 2870
1 26164 66.643 1743650
1 (noise) 26164 0.213 5575
2 48848 84.999 4152046
2 (noise) 48848 0.086 4192
5 48253 57.989 2798158
5 (noise) 48253 0.023 1110

Fitting a Straight Line

Group 14 IMAGE J RESULTS.png