LAB 5 WRITE-UP
SYBR Green Dye
The process of the lab was to use the green dye to test if the green dye and the DNA shows color when placed in the fluorimeter device. The first step was to place 80uL drop of SYBR Green Dye I solution on the first 2 clear circles in the middle of the slide. The second step was to place 80uL of sample calibration solution on top of the SYBR Green dye. The next step was to adjust the slide so that the blue light can go through the center of the drop of the SYBR Green Dye.
The fluorimeter device acts as the main part of this lab and it is very vital. The flourimeter acts as the base to where it illuminates blue light through the solution. The fluorimeter heavily depends on the smart phone to capture the solution and the light that goes through it. The flourimeter measures the amount of light that illuminates through the SYBR Green Dye and the DNA
How the Fluorescence Technique Works
The technique was to use the steps that were given and repeat it until there is five possible measurement position on the slides. The lab required a sample calibration of solution on top of the SYBR Green Dye at least five times to gather data.
Smart Phone Camera Settings
- Type of Smartphone:iPhone 5s
- Flash: Off
- ISO setting:800
- White Balance: Auto
- Exposure: Auto
- Saturation: Auto
- Contrast: Auto
First make sure the fluorimeter is on a flat surface. Then use the cradle to hold the smartphone in place. The camera must be at a right angle from the slide in order to take a picture of the drop sideways. Adjust the height of the fluorimeter by turning the nob holding the plastic tray in place. This will help the camera take a better angle of the drop. The distance between the smartphone and the fluorimeter is then measured with a regular ruler. This distance is held constant in order to get a constant yet accurate image of each individual drop. If the distances are different for each trial then there will be a significant difference from one image to the next due to differences in the light collected. The smartphone should be at least 4cm away from the drop. If not, adjust the smartphone so that the smartphone with its cradle is as close to the first two rows of the slide without taking a blurry image. Once the smartphone is adjusted, take a standard ruler and measure the distance between the smartphone and fluorimeter. It is important not to move the camera, cradle, or fluorimeter, because this is the official position that the smartphone needs to be at throughout the whole experiment.
- Distance between the smart phone cradle and drop = 5cm
Solutions Used for Calibration
Placing Samples onto the Fluorimeter
Step 1) First use gloves in order to adjust the fluorimeter. Turn on the fluorimeter, place it on a smooth surface (preferably a table with a lot of room), and take a glass slide (smooth side down) and place it in the fluorimeter.
Step 2) Set the camera in the appropriate position as described above. Make sure the fluorimeter is adjusted to get a clear view of the slide that is sideways as well as nearly edge-on. Also set a 3 second timer on the camera ready to take a
picture of the drop.
Step 3) Using the plastic pipette, place a 80 microliter drop of SYBR Green I in the middle of the two first clear circles, so that it makes a beach ball-like sphere.
Step 4) Change the tip of the plastic pipetter and use it to add 80 microliters of calf thymus (or water blank) solutions onto that same sphere of SYBR Green I. The beach-ball like sphere is now considered a "drop" or a "sample"
Step 5) Move the slide in order to align the drop. Make sure that the blue LED light is focused on the drop and through the other side of the drop that has a black fiber optic fitting.
Step 6) Cover the fluormeter with a light box, and make sure the camera timer is on in the smartphone.
Step 7) Have the camera take two images of the drop.
Step 8) Remove the box after two images have been successfully taken.
Step 9) Use pipette to remove 160 microliter drop from the glass. Then move slide to the next position and continue procedure for the remaining Calf Thymus DNA solution concentrations of 0.25, 0.5. 1, 2, 5 (microg/mL).
Representative Images of Samples
Image of no DNA in SYBR Green I
Image of 5 microg/mL of DNA in SYBR Green I
Image J Values for All Samples
Fitting a Straight Line