Open PCR Machine Engineer/ Team Leader
Name: Joe Sansone
LAB 2 WRITE-UP
SYBR Green Dye
SYBR Green dye is a dye used as an indicator in molecular biology. It emits green light when bound to double helix DNA and illuminated with blue light. Source: http://en.wikipedia.org/wiki/SYBR_Green_I
[A description of the single-drop fluorimeter device. Add a PHOTO for bonus points]
How the Fluorescence Technique Works
[In your own words, a summary of the information from page 9 of the worksheet]
Smart Phone Camera Settings
- The smartphone used for the camera was the Android Google Nexus 4. An app named Camera Self-Timer was installed to create a window in which the camera can shoot a picture in which the fluorimeter could then be covered in darkness for the most accurate results.
- Flash: No flash was used
- ISO setting: Unknown (could not be altered)
- White Balance: White balance was set on auto
- Exposure: Exposure was set on auto
- Saturation: Saturation was set on high
- Contrast: Contrast was set on low
The camera was placed on a cradle that was in approximately equal height of the fluorimeter. If the cradle and camera needed to be taller in order to be of equal height to the fluorimeter, the cradle was then placed on a stacked glass case until the camera was parallel. The distance from the cradle to the fluorimeter was about 7 cm. After creating a solution for calibration the camera was then set on a self-timer of 10 seconds and then the fluorimeter was encased in a box and covered for complete darkness for the most accurate results. After the beep indicating that the picture was taken the cycle was then complete and the process was then repeated for each solution.
- Distance between the smart phone cradle and drop = 7cm
Solutions Used for Calibration
| Calf Thymus DNA Solution (microg/mL) || Volume 2X DNA Solution (uL) || Volume SYBR GREEN I Solution (uL)|| Fina DNA concentration in PicoGreen 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
- Step 1: A micro pipette was obtained and 80uL of the SYBR GREEN I was dispensed in the center of the first two rows of the slide. Once done 80uL of the desired solution (calf thymus at various concentrations or just water) was added to the mixture on the slide.
- Step two: After the desired mixture was placed onto the slide it was then positioned so that the droplet was centered with the small opening on the apparatus where the blue LED light originates from.
- Step three: After collecting the qualitative data with picture captures, the sample was removed from the slide and discarded appropriately. Then the above steps were repeated for all concentrations of DNA making sure not cross contaminate the mixtures by using different slide rows for each trial.
- Step four: Once all experimental trials were done the work station was cleaned by discarding the slides in the appropriate sharps container as well as dispensing the DNA solutions appropriately. The data was compiled and tabulated for further analysis.
Representative Images of Samples
[Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA]
[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 [See worksheet page 5]
| DNA final concentration (ng/mL)|| Ave. Mean|| Ave. IntDen
| 2.5 || row 2 cell 2 || row 2 cell 3
| 1 || row 3 cell 2 || row 3 cell 3
| 0.5 || row 2 cell 2 || row 2 cell 3
| 0.25|| row 2 cell 2 || row 2 cell 3
| 0.125 || row 2 cell 2 || row 2 cell 3
| 0 || row 2 cell 2 || row 2 cell 3
[Add more rows as needed]
Fitting a Straight Line
[Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 8]